Anti-HtrA1 antibodies and methods of use thereof

ABSTRACT

The present invention provides anti-HtrA1 antibodies (including bispecific anti-HtrA1 anti-Factor D antibodies) and methods of making and using the same, for example, in methods of treating HtrA1-associated disorders, ocular disorders, and/or complement-associated disorders.

FIELD OF THE INVENTION

The invention relates generally to anti-HtrA1 antibodies and methods ofusing the same.

BACKGROUND OF THE INVENTION

The serine protease HtrA serine peptidase 1 (HtrA1) (PRSS11; Clan PA,family 51) belongs to an evolutionarily conserved family of HtrAproteins. In humans, HtrA1, HtrA3, and HtrA4 share the same domainarchitecture: an N-terminal IGFBP-like module and a Kazal-like module, aprotease domain with trypsin-like fold, and a C-terminal PDZ domain. Thephysiological relevance of HtrA1 has been firmly established by theidentification of human loss-of-function mutations causing familialischemic cerebral small-vessel disease. The molecular mechanism involvesdeficient TGFβ inhibition by HtrA1 resulting in increased TGFβsignaling. Dysregulated TGFβ signaling by aberrant HtrA1 expression mayalso contribute to arthritic disease, perhaps in conjunction withHtrA1-mediated degradation of various extracellular matrix components,or indirectly via up-regulation of matrix metalloproteases. In addition,human genetic studies identified a strong correlation betweenprogression of age-related macular degeneration (AMD) and a SNP in theHtrA1 promoter region which results in increased HtrA1 transcript levels(see, e.g., Dewan et al., Science 314:989-992, 2006 and Yang et al.,Science 314:992-993, 2006).

AMD is a progressive chronic disease of the central retina withsignificant consequences for visual acuity. Late forms of the diseaseare the leading cause of vision loss in industrialized countries. Forthe Caucasian population ≥40 years of age, the prevalence of early AMDis estimated at about 6.8% and advanced AMD at about 1.5%. Theprevalence of late AMD increases dramatically with age rising to about11.8% after 80 years of age. Two types of AMD exist, non-exudative (dry)and exudative (wet) AMD. The more common dry AMD involves atrophic andhypertrophic changes in the retinal pigment epithelium (RPE) underlyingthe central retina (macula) as well as deposits (drusen) on the RPE.Advanced dry AMD can result in significant retinal damage, includinggeographic atrophy (GA), with irreversible vision loss. Moreover,patients with dry AMD can progress to the wet form, in which abnormalblood vessels called choroidal neovascular membranes (CNVMs) developunder the retina, leak fluid and blood, and ultimately cause a blindingdisciform scar in and under the retina.

There remains a need for anti-HtrA1 antibodies with improved properties,such as binding affinity, stability, and inhibitory (blocking) activity,as well as therapeutic and diagnostic uses thereof.

SUMMARY OF THE INVENTION

The present invention provides anti-HtrA1 antibodies and methods ofusing the same for therapeutic and diagnostic purposes. The anti-HtrA1antibodies of the invention are highly potent and have a high bindingaffinity for HtrA1. The improved properties of the antibodies of theinvention make them suitable for use in therapy.

In one aspect, the invention encompasses an isolated antibody thatspecifically binds to an HtrA1 epitope, where the HtrA1 epitopecomprises at least one amino acid of the HtrA1 protein selected from thegroup consisting of Arg190, Leu192, Pro193, Phe194, and Arg197, wherethe amino acid numbering refers to the numbering for the human HtrA1precursor protein.

In one embodiment, the HtrA1 epitope comprises at least one amino acidof the HtrA1 protein selected from the group consisting of Leu192,Pro193, and Arg197.

In another embodiment, the HtrA1 epitope comprises at least two aminoacids of the HtrA1 protein selected from the group consisting of Leu192,Pro193, and Arg197.

In a particular embodiment, the HtrA1 epitope comprises the HtrA1 aminoacids Leu192, Pro193, and Arg197.

In another embodiment, the HtrA1 epitope comprises the HtrA1 amino acidsArg190, Leu192, Pro193, and Arg197.

In an additional embodiment, the HtrA1 epitope comprises the HtrA1 aminoacids Arg190, Leu192, Pro193, Phe194, and Arg197.

In one aspect, the invention features an isolated antibody thatspecifically binds human HtrA serine peptidase 1 (HtrA1) with a KD ofabout 550 pM or lower. In some embodiments, the antibody specificallybinds human HtrA1 with a KD between about 40 pM and about 550 pM. Insome embodiments, the antibody specifically binds human HtrA1 with a KDbetween about 40 pM and about 250 pM. In some embodiments, the antibodyspecifically binds human HtrA1 with a KD between about 50 pM and about125 pM. In some embodiments, the antibody specifically binds human HtrA1with a KD of about 110 pM. In some embodiments, the antibodyspecifically binds human HtrA1 with a KD of about 60 pM. In someembodiments, the KD is measured by surface plasmon resonance (SPR)(e.g., BIACORE® SPR). In some embodiments, the SPR is performed asdescribed herein (e.g., in the Examples section).

In some embodiments, any one of the preceding antibodies is capable ofinhibiting the activity of HtrA1. In some embodiments, the antibodyinhibits the activity of the protease domain of human HtrA1(huHtrA1-PD)with a 50% inhibitory concentration (IC50) of 1.5 nM or lower. In someembodiments, the antibody inhibits the activity of huHtrA1-PD with anIC50 of 0.25 nM to about 0.5 nM. In some embodiments, the antibodyinhibits the activity of huHtrA1-PD with an IC50 of about 0.3 nM. Insome embodiments, the inhibitory activity is an in vitro FRET-basedblocking assay measurement. In some embodiments, the in vitro FRET-basedblocking assay comprises cleavage of an H2-Opt probe (e.g., SEQ ID NO:152). In some embodiments, the in vitro FRET-based blocking assay isperformed as described herein (e.g., in the Examples).

In some embodiments of the above aspect, the antibody comprises abinding domain comprising: (a) an HVR-H1 comprising the amino acidsequence of DSEX₁H (SEQ ID NO: 1), wherein X₁ is Met or Leu; (b) anHVR-H2 comprising the amino acid sequence of GVDPETX₂GAAYNQKFKG (SEQ IDNO: 2), wherein X₂ is Glu or Asp; and (c) an HVR-H3 comprising the aminoacid sequence of GYDYDYALDY (SEQ ID NO: 3). In some embodiments, theantibody comprises a binding domain comprising: (a) an HVR-H1 comprisingthe amino acid sequence of DSEMH (SEQ ID NO: 7); (b) an HVR-H2comprising the amino acid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8);and (c) an HVR-H3 comprising the amino acid sequence of GYDYDYALDY (SEQID NO: 3). In some embodiments, the antibody further comprises: (a) anFR-H1 comprising the amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASGYX₁FX₂ (SEQ ID NO: 12), wherein X₁ is Lys orThr and X₂ is Thr, Lys, or Arg; (b) an FR-H2 comprising the amino acidsequence of WVRQAPGQGLEWIG (SEQ ID NO: 13); (c) an FR-H3 comprising theamino acid sequence of RATITRDTSTSTAYLELSSLRSEDTAVYYCTR (SEQ ID NO: 14);and (d) an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQID NO: 15). In some embodiments, the antibody further comprises: (a) anFR-H1 comprising the amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASGYKFT (SEQ ID NO: 16); (b) an FR-H2 comprisingthe amino acid sequence of WVRQAPGQGLEWIG (SEQ ID NO: 13); (c) an FR-H3comprising the amino acid sequence of RATITRDTSTSTAYLELSSLRSEDTAVYYCTR(SEQ ID NO: 14); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 15).

In some embodiments of the above aspect, the binding domain furthercomprises: (a) an HVR-L1 comprising the amino acid sequence ofRASSSVX₃FIH (SEQ ID NO: 4), wherein X₃ is Glu or Asn; (b) an HVR-L2comprising the amino acid sequence of ATSX₄LAS (SEQ ID NO: 5), whereinX₄ is Asn, His or Glu; and (c) an HVR-L3 comprising the amino acidsequence of QQWX₅SX₆PWT (SEQ ID NO: 6), wherein X₅ is Ser or Tyr and X₆is Ala or Asn. In some embodiments, the binding domain furthercomprises: (a) an HVR-L1 comprising the amino acid sequence ofRASSSVEFIH (SEQ ID NO: 9); (b) an HVR-L2 comprising the amino acidsequence of ATSNLAS (SEQ ID NO: 10); and (c) an HVR-L3 comprising theamino acid sequence of QQWSSAPWT (SEQ ID NO: 11). In some embodiments,the antibody further comprises: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKPLIS (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a heavy chain variable (VH) domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 21; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 22; or (c) a VH domain as in(a) and a VL domain as in (b). In some embodiments, the VH domainfurther comprises: (a) an FR-H1 comprising the amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASGYKFT (SEQ ID NO: 16); (b) an FR-H2 comprisingthe amino acid sequence of WVRQAPGQGLEWIG (SEQ ID NO: 13); (c) an FR-H3comprising the amino acid sequence of RATITRDTSTSTAYLELSSLRSEDTAVYYCTR(SEQ ID NO: 14); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 15). In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO: 21. In some embodiments,the VL domain further comprises: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKPLIS (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome embodiments, the VL domain comprises the amino acid sequence of SEQID NO: 22.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising the following six HVRs: (a) an HVR-H1 comprising theamino acid sequence of DSEX₁H (SEQ ID NO: 1), wherein X₁ is Met or Leu;(b) an HVR-H2 comprising the amino acid sequence of GVDPETX₂GAAYNQKFKG(SEQ ID NO: 2), wherein X₂ is Glu or Asp; (c) an HVR-H3 comprising theamino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASSSVX₃FIH (SEQ ID NO: 4),wherein X₃ is Glu or Asn; (e) an HVR-L2 comprising the amino acidsequence of ATSX₄LAS (SEQ ID NO: 5), wherein X₄ is Asn, His or Glu; and(f) an HVR-L3 comprising the amino acid sequence of QQWX₅SX₆PWT (SEQ IDNO: 6), wherein X₅ is Ser or Tyr and X₆ is Ala or Asn. In someembodiments, the binding domain comprises the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of DSEMH (SEQ ID NO: 7); (b)an HVR-H2 comprising the amino acid sequence of GVDPETEGAAYNQKFKG (SEQID NO: 8); (c) an HVR-H3 comprising the amino acid sequence ofGYDYDYALDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASSSVEFIH (SEQ ID NO: 9); (e) an HVR-L2 comprising theamino acid sequence of ATSNLAS (SEQ ID NO: 10); and (f) an HVR-L3comprising the amino acid sequence of QQWSSAPWT (SEQ ID NO: 11).

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a VH domain comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 21; (b) a VL domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 22;or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the VH domain further comprises: (a) an FR-H1 comprisingthe amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYKFT (SEQ ID NO:16); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGQGLEWIG(SEQ ID NO: 13); (c) an FR-H3 comprising the amino acid sequence ofRATITRDTSTSTAYLELSSLRSEDTAVYYCTR (SEQ ID NO: 14); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15). Insome embodiments, the VH domain comprises the amino acid sequence of SEQID NO: 21. In some embodiments, the VL domain further comprises: (a) anFR-L1 comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQID NO: 17); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKPLIS (SEQ ID NO: 18); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 19); and (d) anFR-L4 comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).In some embodiments, the VL domain comprises the amino acid sequence ofSEQ ID NO: 22.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a VH domain comprising an amino acid sequencehaving at least 99% sequence identity to the amino acid sequence of SEQID NO: 21 and (b) a VL domain comprising an amino acid sequence havingat least 99% sequence identity to the amino acid sequence of SEQ ID NO:22.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a heavy chain variable (VH) domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 23; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 24; or (c) a VH domain as in(a) and a VL domain as in (b). In some embodiments, the VH domainfurther comprises: (a) an FR-H1 comprising the amino acid sequence ofQVQLQQSGAELVRPGASVTLSCKASGYTFT (SEQ ID NO: 24); (b) an FR-H2 comprisingthe amino acid sequence of WVKQTPVHGLEWIG (SEQ ID NO: 25); (c) an FR-H3comprising the amino acid sequence of KATLTADKSSSTAYMELRSLTSEDSAVYYCTR(SEQ ID NO: 26); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTSVTVSS (SEQ ID NO: 27). In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO: 23. In some embodiments,the VL domain further comprises: (a) an FR-L1 comprising the amino acidsequence of NIVVTQSPASLAVSLGQRATISC (SEQ ID NO: 29); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 30);(c) an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 31); and (d) an FR-L4comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 32). Insome embodiments, the VL domain comprises the amino acid sequence of SEQID NO: 24.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a VH domain comprising an amino acid sequencehaving at least 99% sequence identity to the amino acid sequence of SEQID NO: 23 and (b) a VL domain comprising an amino acid sequence havingat least 99% sequence identity to the amino acid sequence of SEQ ID NO:24.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising the following six HVRs: (a) an HVR-H1 comprising theamino acid sequence of SYIMS (SEQ ID NO: 39); (b) an HVR-H2 comprisingthe amino acid sequence of YISNGGGTTYYSDTIKG (SEQ ID NO: 40); (c) anHVR-H3 comprising the amino acid sequence of QNFRSDGSSMDY (SEQ ID NO:41); (d) an HVR-L1 comprising the amino acid sequence of RASESVDSYGKSFMH(SEQ ID NO: 42); (e) an HVR-L2 comprising the amino acid sequence ofLASKLES (SEQ ID NO: 43); and (f) an HVR-L3 comprising the amino acidsequence of QQNNEDPYT (SEQ ID NO: 44).

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a VH domain comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 45; (b) a VL domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 46;or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the VH domain further comprises: (a) an FR-H1 comprisingthe amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:47); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA(SEQ ID NO: 48); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 49); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 50). Insome embodiments, the VH domain comprises the amino acid sequence of SEQID NO: 45. In some embodiments, the VL domain further comprises: (a) anFR-L1 comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINC (SEQID NO: 51); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGQPPKLLIY (SEQ ID NO: 52); (c) an FR-L3 comprising the amino acidsequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 53); and (d) anFR-L4 comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 54).In some embodiments, the VL domain comprises the amino acid sequence ofSEQ ID NO: 46.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a VH domain comprising an amino acid sequencehaving at least 99% sequence identity to the amino acid sequence of SEQID NO: 45 and (b) a VL domain comprising an amino acid sequence havingat least 99% sequence identity to the amino acid sequence of SEQ ID NO:46.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a heavy chain variable (VH) domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 55; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 56; or (c) a VH domain as in(a) and a VL domain as in (b). In some embodiments, the VH domainfurther comprises: (a) an FR-H1 comprising the amino acid sequence ofEVKLVESGGGLVEPGGSLKLACVASGFTFS (SEQ ID NO: 57); (b) an FR-H2 comprisingthe amino acid sequence of WVRQTPEKRLEWVA (SEQ ID NO: 58); (c) an FR-H3comprising the amino acid sequence of RFTISRDNAKNTLYLQMSTLKSEDTAIYFCAR(SEQ ID NO: 59); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTAVTVSS (SEQ ID NO: 60). In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO: 55. In some embodiments,the VL domain further comprises: (a) an FR-L1 comprising the amino acidsequence of NIVVTQSPASLAVSLGQRATISC (SEQ ID NO: 61); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 62);(c) an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 63); and (d) an FR-L4comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 64). Insome embodiments, the VL domain comprises the amino acid sequence of SEQID NO: 56.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a VH domain comprising an amino acid sequencehaving at least 99% sequence identity to the amino acid sequence of SEQID NO: 55 and (b) a VL domain comprising an amino acid sequence havingat least 99% sequence identity to the amino acid sequence of SEQ ID NO:56.

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a heavy chain variable (VH) domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 65; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 66; or (c) a VH domain as in(a) and a VL domain as in (b).

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a heavy chain variable (VH) domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 67; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 68; or (c) a VH domain as in(a) and a VL domain as in (b).

In another aspect, the invention features an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising (a) a heavy chain variable (VH) domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 69; (b) a light chain variable (VL) domaincomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 70; or (c) a VH domain as in(a) and a VL domain as in (b).

In some embodiments of any of the preceding aspects, the antibody ismonoclonal, human, humanized, or chimeric. In particular embodiments,the antibody is monoclonal, humanized, or chimeric.

In some embodiments of any of the preceding aspects, the antibody is anantibody fragment that binds to HtrA1. In some embodiments, the antibodyfragment is selected from the group consisting of Fab, Fab′-SH, Fv,scFV, and (Fab′)₂ fragments. In some embodiments, the antibody fragmentis an Fab. In some embodiments, the Fab comprises a truncation in thehinge region of the heavy chain constant region. In some embodiments,the Fab comprises a truncation in the upper hinge of the heavy chainconstant region. In some embodiments, the heavy chain constant regionterminates at position 221 (EU numbering). In some embodiments, theamino acid residue at position 221 is an aspartic acid (Asp) residue. Insome embodiments, the heavy chain constant region comprises the aminoacid sequence of SEQ ID NO: 156. In some embodiments, the antibodycomprises the heavy chain amino acid sequence of SEQ ID NO: 160. In someembodiments, the antibody comprises the light chain amino acid sequenceof SEQ ID NO: 159. In some embodiments, the antibody comprises the heavychain amino acid sequence of SEQ ID NO: 160 and the light chain aminoacid sequence of SEQ ID NO: 159.

In some embodiments, the Fab is an IgG1 Fab.

In some embodiments of any of the preceding aspects, the antibody is afull-length antibody. In some embodiments, the antibody is an IgGantibody. In some embodiments, the antibody is a monospecific antibody.

In some embodiments of any of the preceding aspects, the antibody is abispecific antibody. In some embodiments, the bispecific antibodycomprises a second binding domain that binds to Factor D. In someembodiments, the second binding domain comprises the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ IDNO: 109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp or Glu; (c) anHVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ ID NO: 111),wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the amino acidsequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp or Ser,X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprising theamino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu. In some embodiments, the second binding domaincomprises the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2 comprisingthe amino acid sequence of WINTYTGETTYADDFKG (SEQ ID NO: 115); (c) anHVR-H3 comprising the amino acid sequence of EGGVNN (SEQ ID NO: 116);(d) an HVR-L1 comprising the amino acid sequence of ITSTDIDDDMN (SEQ IDNO: 117); (e) an HVR-L2 comprising the amino acid sequence of GGNTLRP(SEQ ID NO: 113); and (f) an HVR-L3 comprising the amino acid sequenceof LQSDSLPYT (SEQ ID NO: 118). In some embodiments, the second bindingdomain comprises (a) a VH domain comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO: 119; (b) a VL domain comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:120; or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the VH domain comprises the amino acid sequence of SEQ IDNO: 119. In some embodiments, the VL domain comprises the amino acidsequence of SEQ ID NO: 120.

In another aspect, the invention features an isolated antibody thatspecifically binds both HtrA1 and Factor D, wherein the antibodycomprises a first binding domain that specifically binds HtrA1comprising the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of DSEMH (SEQ ID NO: 7); (b) an HVR-H2 comprising theamino acid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) an HVR-H3comprising the amino acid sequence of GYDYDYALDY (SEQ ID NO: 3), (d) anHVR-L1 comprising the amino acid sequence of RASSSVEFIH (SEQ ID NO: 9);(e) an HVR-L2 comprising the amino acid sequence of ATSNLAS (SEQ ID NO:10); and (1) an HVR-L3 comprising the amino acid sequence of QQWSAPWT(SEQ ID NO: 11); and a second binding domain that specifically bindsFactor D comprising the following six HVRs: (a) an HVR-H1 comprising theamino acid sequence of GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2comprising the amino acid sequence of WINTYTGETTYADDFKG (SEQ ID NO:115); (c) an HVR-H3 comprising the amino acid sequence of EGGVNN (SEQ IDNO: 116); (d) an HVR-L1 comprising the amino acid sequence ofITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2 comprising the amino acidsequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3 comprising theamino acid sequence of LQSDSLPYT (SEQ ID NO: 118).

In another aspect, the invention features an isolated antibody thatspecifically binds both HtrA1 and Factor D, wherein the antibodycomprises a first binding domain that specifically binds HtrA1comprising (a) a VH domain comprising an amino acid sequence having atleast 99% sequence identity to the amino acid sequence of SEQ ID NO: 21and (b) a VL domain comprising an amino acid sequence having at least99% sequence identity to the amino acid sequence of SEQ ID NO: 22, and asecond binding domain that specifically binds Factor D comprising (a) aVH domain comprising an amino acid sequence having at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 119 and (b) a VLdomain comprising an amino acid sequence having at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 120.

In some embodiments of the above aspects, the invention encompasses anisolated antibody that specifically binds to an HtrA1 epitope, where theHtrA1 epitope comprises at least one amino acid of the HtrA1 proteinselected from the group consisting of Arg190, Leu192, Pro193, Phe194,and Arg197, where the amino acid numbering refers to the numbering forthe human HtrA1 precursor protein.

In one embodiment, the HtrA1 epitope comprises at least one amino acidof the HtrA1 protein selected from the group consisting of Leu192,Pro193, and Arg197.

In a particular embodiment, the HtrA1 epitope comprises the HtrA1 aminoacids Leu192, Pro193, and Arg197.

In another embodiment, the HtrA1 epitope comprises the HtrA1 amino acidsArg190, Leu192, Pro193, and Arg197.

In an additional embodiment, the HtrA1 epitope comprises the HtrA1 aminoacids Arg190, Leu2, Pro193, Phe194, and Arg197.

In another aspect, the invention features an isolated nucleic acidencoding any of the antibodies described herein. In another aspect, theinvention features a vector (e.g., an expression vector) comprising theisolated nucleic acid for expressing the antibody. In another aspect,the invention features host cells comprising the preceding nucleic acidsand/or vectors. In some embodiments, the host cell is a mammalian cell.In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO)cell or a 293 cell. In some embodiments, the host cell is a prokaryoticcell. In some embodiments, the prokaryotic cell is E. coli.

In another aspect, the invention features a method of producing any ofthe antibodies described herein, the method comprising culturing a hostcell that comprises any of the preceding vectors (e.g., expressionvectors) in a culture medium. In some embodiments, the method furthercomprises recovering the antibody from the host cell or the culturemedium.

In another aspect, the invention features a composition comprising anyone of the preceding antibodies. In some embodiments, the compositionfurther comprises a pharmaceutically acceptable carrier, excipient, ordiluent. In some embodiments, the composition is a pharmaceuticalcomposition. In some embodiments, the pharmaceutical composition islyophilized. In some embodiments, the composition further comprises aFactor D binding antagonist. In some embodiments, the Factor D bindingantagonist is an anti-Factor D antibody or an antigen-binding fragmentthereof. In some embodiments, the antigen-binding fragment is an Fab oran (Fab′)₂. In some embodiments, the anti-Factor D antibody orantigen-binding fragment thereof comprises the following six HVRs: (a)an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ ID NO:109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp or Glu; (c) anHVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ ID NO: 111),wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the amino acidsequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp or Ser,X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprising theamino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu. In some embodiments, the anti-Factor Dantibody or antigen-binding fragment thereof comprises the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN(SEQ ID NO: 109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYADDFKG (SEQ ID NO: 115); (c) an HVR-H3 comprising the aminoacid sequence of EGGVNN (SEQ ID NO: 116); (d) an HVR-L1 comprising theamino acid sequence of ITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2comprising the amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f)an HVR-L3 comprising the amino acid sequence of LQSDSLPYT (SEQ ID NO:118). In some embodiments, the anti-Factor D antibody or antigen-bindingfragment thereof comprises (a) a VH domain comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 119; (b) a VL domain comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 120; or (c) a VH domain as in (a) and a VL domainas in (b). In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO: 119. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 120. In some embodiments, theanti-Factor D antigen-binding antibody fragment is lampalizumab.

In another aspect, the invention encompasses a combination therapycomprising any of the preceding anti-HtrA1 antibodies and a Factor Dantagonist. In a particular embodiment, the Factor D antagonist is ananti-Factor D antibody. In a particular embodiment, the Factor Dantagonist is lampalizumab. In a particular embodiment, the anti-FactorD antagonist is administered sequentially.

In some aspects, any one of the preceding antibodies can be used as amedicament.

In some aspects, any one of the preceding antibodies can be used intreating an HtrA1-associated disorder or an ocular disorder. In someembodiments, the HtrA1-associated disorder or the ocular disorder isage-related macular degeneration (AMD), diabetic retinopathy,retinopathy of prematurity, or polypoidal choroidal vasculopathy. Insome embodiments, the HtrA1-associated disorder or the ocular disorderis AMD. In some embodiments, the AMD is early dry AMD, intermediate dryAMD, or advanced dry AMD. In some embodiments, the advanced dry AMD isgeographic atrophy.

In some aspects, any one of the preceding antibodies can be used in themanufacture of a medicament for treating an HtrA1-associated disorder oran ocular disorder. In some embodiments, the HtrA1-associated disorderor the ocular disorder is AMD, diabetic retinopathy, retinopathy ofprematurity, or polypoidal choroidal vasculopathy. In some embodiments,the HtrA1-associated disorder or the ocular disorder is AMD. In someembodiments, the AMD is early dry AMD, intermediate dry AMD, or advanceddry AMD. In some embodiments, the advanced dry AMD is geographicatrophy. In some embodiments, the medicament is formulated for use incombination with a Factor D binding antagonist. In some embodiments, theFactor D binding antagonist is an anti-Factor D antibody or anantigen-binding fragment thereof. In some embodiments, theantigen-binding fragment is an Fab or an (Fab′)₂. In some embodiments,the the anti-Factor D antibody or antigen-binding fragment thereofcomprises the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2 comprisingthe amino acid sequence of WINTYTGETTYAX₁DFKG (SEQ ID NO: 110), whereinX₁ is Asp or Glu; (c) an HVR-H3 comprising the amino acid sequence ofEGGVX₁N (SEQ ID NO: 111), wherein X₁ is Asn or Ser; (d) an HVR-L1comprising the amino acid sequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112),wherein X₁ is Asp or Ser, X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) anHVR-L2 comprising the amino acid sequence of GGNTLRP (SEQ ID NO: 113);and (f) an HVR-L3 comprising the amino acid sequence of LQSX₁SLPYT (SEQID NO: 114), wherein X₁ is Asp or Glu. In some embodiments, theanti-Factor D antibody or antigen-binding fragment thereof comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofGYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2 comprising the amino acidsequence of WINTYTGETTYADDFKG (SEQ ID NO: 115); (c) an HVR-H3 comprisingthe amino acid sequence of EGGVNN (SEQ ID NO: 116); (d) an HVR-L1comprising the amino acid sequence of ITSTDIDDDMN (SEQ ID NO: 117); (e)an HVR-L2 comprising the amino acid sequence of GGNTLRP (SEQ ID NO:113); and (f) an HVR-L3 comprising the amino acid sequence of LQSDSLPYT(SEQ ID NO: 118). In some embodiments, the anti-Factor D antibody orantigen-binding fragment thereof comprises (a) a VH domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 119; (b) a VL domain comprising an aminoacid sequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 120; or (c) a VH domain as in (a) and a VL domainas in (b). In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO: 119. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 120. In some embodiments, theanti-Factor D antigen-binding fragment is lampalizumab.

In another aspect, the invention features a method of treating anHtrA1-associated disorder or an ocular disorder in a subject in needthereof, the method comprising administering a therapeutically effectiveamount of the antibody of any one of the preceding antibodies. In someembodiments, the HtrA1-associated disorder or the ocular disorder isAMD, diabetic retinopathy, retinopathy of prematurity, or polypoidalchoroidal vasculopathy. In some embodiments, the HtrA1-associateddisorder or the ocular disorder is AMD. In some embodiments, the AMD isearly dry AMD, intermediate dry AMD, or advanced dry AMD. In someembodiments, the advanced dry AMD is geographic atrophy. In someembodiments, the method further comprises administering a Factor Dbinding antagonist.

In another aspect, the invention features a method for inhibitingretinal or photoreceptor cell degeneration in a subject, the methodcomprising administering to the subject an effective amount of any oneof the preceding antibodies, thereby inhibiting retinal or photoreceptorcell degeneration.

In another aspect, the invention features a method for inhibiting HtrA1serine protease activity in an eye of a subject, the method comprisingadministering to the subject an effective amount of any one of thepreceding antibodies, thereby inhibiting HtrA1 serine protease activityin the eye. In some embodiments, the method further comprisesadministering a Factor D binding antagonist.

In another aspect, the invention features a method of treating anHtrA1-associated disorder or a complement-associated disorder in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of an HtrA1 bindingantagonist and a Factor D binding antagonist. In some embodiments, theHtrA1-associated disorder or the complement-associated disorder is anocular disorder. In some embodiments, the ocular disorder is selectedfrom the group consisting of AMD, diabetic retinopathy, choroidalneovascularization (CNV), uveitis, diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, centralretinal vein occlusion, corneal vascularization, and retinalneovascularization. In some embodiments, the ocular disorder is AMD. Insome embodiments, the AMD is early dry AMD, intermediate dry AMD, oradvanced dry AMD. In some embodiments, the advanced dry AMD isgeographic atrophy. In some embodiments, the HtrA1 binding antagonist isan anti-HtrA1 antibody or an antigen-binding fragment thereof. In someembodiments, the antigen-binding fragment is selected from the groupconsisting of Fab, Fab′-SH, Fv, scFV, and (Fab′)₂ fragments. In someembodiments, the antigen-binding fragment is an Fab. In someembodiments, the Fab comprises a truncation in the upper hinge of theheavy chain constant region. In some embodiments, the heavy chainconstant region terminates at position 221 (EU numbering). In someembodiments, the amino acid residue at position 221 is an aspartic acid(Asp) residue. In some embodiments, the heavy chain constant regioncomprises the amino acid sequence of SEQ ID NO: 156. In someembodiments, the antibody comprises the heavy chain amino acid sequenceof SEQ ID NO: 160. In some embodiments, the antibody comprises the lightchain amino acid sequence of SEQ ID NO: 159. In some embodiments, theantibody comprises the heavy chain amino acid sequence of SEQ ID NO: 160and the light chain amino acid sequence of SEQ ID NO: 159. In someembodiments, the Fab is an IgG1 Fab.

In another aspect, the invention features a method of treating anHtrA1-associated disorder or a complement-associated disorder in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of any one of the precedingantibodies and a therapeutically effective amount of a Factor D bindingantagonist.

In some embodiments of any of the preceding aspects, the Factor Dbinding antagonist is an anti-Factor D antibody or an antigen-bindingfragment thereof. In some embodiments, the antigen-binding fragment isan Fab or an (Fab′)₂. In some embodiments, the anti-Factor D antibody orantigen-binding fragment thereof comprises the following six HVRs: (a)an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ ID NO:109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp or Glu; (c) anHVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ ID NO: 111),wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the amino acidsequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp or Ser,X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprising theamino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu. In some embodiments, the anti-Factor Dantibody or antigen-binding fragment thereof comprises the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN(SEQ ID NO: 109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYADDFKG (SEQ ID NO: 115); (c) an HVR-H3 comprising the aminoacid sequence of EGGVNN (SEQ ID NO: 116); (d) an HVR-L1 comprising theamino acid sequence of ITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2comprising the amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f)an HVR-L3 comprising the amino acid sequence of LQSDSLPYT (SEQ ID NO:118). In some embodiments, the anti-Factor D antibody or antigen-bindingfragment thereof comprises (a) a VH domain comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 119; (b) a VL domain comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 120; or (c) a VH domain as in (a) and a VL domainas in (b). In some embodiments, the VH domain comprises the amino acidsequence of SEQ ID NO: 119. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 120. In some embodiments, theanti-Factor D antigen-binding fragment is lampalizumab.

In some embodiments of any of the preceding aspects, theHtrA1-associated disorder or the complement-associated disorder is anocular disorder. In some embodiments, the ocular disorder is selectedfrom the group consisting of AMD, diabetic retinopathy, choroidalneovascularization (CNV), uveitis, diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, centralretinal vein occlusion, corneal vascularization, and retinalneovascularization. In some embodiments, the ocular disorder is AMD. Insome embodiments, the AMD is early dry AMD, intermediate dry AMD, oradvanced dry AMD. In some embodiments, the advanced dry AMD isgeographic atrophy.

In some embodiments of any of the preceding aspects, the antibody isadministered intravitreally, ocularly, intraocularly, juxtasclerally,subtenonly, superchoroidally, topically, intravenously, intramuscularly,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intraperitoneally,peritoneally, intraventricularly, subcutaneously, subconjunctivally,intravesicularly, mucosally, intrapericardially, intraumbilically,intraorbitally, orally, transdermally, by inhalation, by injection, byeye drop, by implantation, by infusion, by continuous infusion, bylocalized perfusion bathing target cells directly, by catheter, bylavage, in cremes, or in lipid compositions. In some embodiments, theantibody is administered intravitreally, ocularly, intraocularly,juxtasclerally, subtenonly, superchoroidally, or topically. In someembodiments, the antibody is administered intravitreally by injection.In some embodiments, the antibody is administered topically by eye dropor ointment. In some embodiments, the antibody is administered by along-acting delivery system. In particular embodiments, the long-actingdelivery system is a PLGA-based solid implant or an implantable portdelivery system.

In some embodiments of any of the preceding aspects, the subject is ahuman.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1B are graphs showing that the majority of enzyme-linkedimmunosorbent assay (ELISA)-positive hybridoma clones showed similarreactivity profiles to both human (hu) and murine (mu) HtrA1 proteasedomain (PD). The graphs show the optical density at 650 nm (OD_(650 nm))for each of the indicated 75 clones. Background signal in this assay was<0.05 OD_(650 nm). The human and murine HtrA1 protease domains share 91%homology.

FIG. 2A is a schematic diagram of a blocking assay used to determine theability of the indicated anti-HtrA1 hybridoma clone supernatants toinhibit HtrA1-PD-mediated cleavage of a BODIPY® FL-labeled fluorescentsubstrate.

FIGS. 2B-2C are graphs showing that 10 anti-HtrA1 hybridoma clonesupernatants markedly inhibited human HtrA1-PD-mediated substratecleavage using the blocking assay described in FIG. 2A and Example 1.The graphs show the average fluorescent signal (milli relativefluorescent units (mRFU)/min) for the hybridoma supernatants from theindicated clones. Antibody YW505.94 (also referred to as “94 IgG,” seeInternational Patent Application Publication No. WO 2013/055998, whichis incorporated herein by reference in its entirety) at 10 μg/ml inconditioned media (CM or cond. medium) served as a positive control toshow that the assay showed no changes due to media and is stable overthe assay time. Buffer or media served as negative controls. FIG. 2Cshows the initial and end results of the assay for buffer, E medium (anutrient-rich medium from CLONACELL™), and conditioned medium. 100%indicates complete inhibition.

FIGS. 3A-3B are graphs showing the ability of the indicated hybridomasupernatants to inhibit cleavage of a fluorescent substrate bymuHtrA1-PD (FIG. 3A) or huHtrA1-PD (FIG. 3B). In FIG. 3A, 40 nM ofmuHtrA1-PD was used in a ratio of 40 μl muHtrA1-PD to 60 μl hybridomasupernatant. In FIG. 3B, 20 nM of huHtrA1-PD was used in a ratio of 40μl huHtrA1-PD to 60 μl hybridoma supernatant. Antibody YW505.94 (“94IgG”) served as a positive control. Buffer and CM served as negativecontrols.

FIG. 4A is a schematic diagram of a FRET-based blocking assay used todetermine the ability of purified anti-HtrA1 antibody clones to inhibitHtrA1-PD-mediated cleavage of a FRET-based substrate, H2-Opt.

FIGS. 4B-4C are graphs showing that purified antibody clones 15H6,19B12, 3A5, 12A5, and 20E2 retained the ability to inhibit murine (FIG.4B) and human (FIG. 4C) HtrA1-PD-mediated substrate cleavage. Theaddition of no antibody (“no ab”) served as a negative control, whileantibody YW505.94 (“94 IgG”) served as a positive control. The purifiedantibodies were added at concentrations of 5 nM, 50 nM, or 500 nM. 15 nMor muHtrA1-PD-Fc was used in the assay presented in FIG. 4B, while 3 nMof huHtrA1-PD-Fc was used in the assay presented in FIG. 4C.

FIGS. 5A-5B are graphs showing that the indicated purified mIgG antibodyclones inhibit full-length human HtrA1 (huHtrA1-FL)-mediated cleavage ofa FRET peptide substrate. The graph shows activity (mRFU/min) as afunction of IgG concentration. huHtrA1-FL was added ata concentration of5 nM. YVV505.94 in IgG format (“IgG94”) and a chimeric variant thereof(“IgG94-ch”) as described in Cifferi et al. (2015) Biochem. J.472(2):169-81 served as positive controls. The half maximal inhibitoryconcentration (IC50) for each antibody clone is shown.

FIGS. 5C-5D are graphs showing that the indicated purified mIgG antibodyclones inhibit muHtrA1-FL-mediated cleavage of a FRET peptide substrate.The graph shows activity (mRFU/min) as a function of IgG concentration.muHtrA1-FL was added at 5 nM. IgG94 and IgG94-ch served as positivecontrols as described in the Figure description for FIGS. 5A and 5B. Thehalf maximal inhibitory concentration (IC50) is shown.

FIG. 6A shows a sequence alignment of amino acid sequences of the heavychain variable region (VH) of antibody clones 19B12, 20E2, 3A5, 12A5,and 15H6.

FIG. 6B shows a sequence alignment of the amino acid sequences of thelight chain variable region (VL) of antibody clones 19B12, 20E2, 3A5,12A5, and 15H6.

FIGS. 7A-7B are graphs showing the results of a FRET-based blockingassay using mIgG antibody clone 15H6 (FIG. 7A) or clone 19B12 (FIG. 7B)purified from hybridoma supernatant (“hyb”) or recombinantly expressedfrom 293 cells (“293”). The graphs plot Wax (mRFU/min) as a function ofantibody concentration (nM). 3 nM of huHtrA1-FL was used in each assay.The IC50 values for the indicated antibody clones are also shown.

FIGS. 8A-8B show sequence alignments of the amino acid sequences of theVL (FIG. 8A) and VH (FIG. 8B) of anti-HtrA1 antibody clones ml5H6,H15H6.v1, H15H6.v2, and h15H6.v2.APEG (also referred to herein as“h15H6.v3”) compared to the human consensus κ1 sequence (FIG. 8A) or VH1sequence (FIG. 8B). HVR sequences are delimited by the denoted boxes foreach of the antibody clones. The HVR sequences according to the Kabatdefinition are underlined. Residues shown in white text in shaded boxesindicates residues that are different between the human consensus VH1sequence and the anti-HtrA1 antibody clones.

FIGS. 9A-9D are graphs showing the results of BIACORE™ surface plasmonresonance (SPR) analysis of binding of m15H6 or h15H6.v1 tostreptavidin-captured biotinylated human or murine HtrA1. Single cyclekinetic analysis was employed. The graphs show response units (RU) as afunction of time (sec). FIG. 9A shows the results from binding of ml5H6Fab to huHtrA1. FIG. 9B shows the results from binding of m15H6 Fab tomuHtrA1. FIG. 9C shows the results from binding of h15H6.v1 to huHtrA1.FIG. 9D shows the results from binding of h15H6.v1 to huHtrA1. TheK_(on), K_(off), and KD determined from each analysis are shown as textinside each graph.

FIGS. 10A-10B are graphs showing the results of phage competition ELISAexperiments for binding of the indicated h15H6.v1 Fab variants to murine(FIG. 10A) or human (FIG. 10B) HtrA1. The graphs show bound phage(OD_(450 nm)) as a function of HtrA1 concentration (nM).

FIGS. 11A-11B are graphs showing the results of BIACORE™ SPR analysiscomparing binding of antibody clone h15H6.v2 and the HVR-L3 LC-W91L andLC-W91Y variants to murine (FIG. 11A) or human (FIG. 11B) HtrA1. Theantibodies used were in Fab format. The K_(on), K_(off), and KDdetermined from each analysis are shown as text inside each graph.

FIG. 12A is a graph showing the results of BIACORE™ SPR analysiscomparing binding of antibody clone h15H6.v2 (parent) and the indicatedvariants at VL position 94 (i.e., LC-N94A LC-P95 (AP), LC-N94E LC-P95(EP), LC-N94Q LC-P95 (QP), and LC-N94S LC-P95 (SP)) to huHtrA1. Theantibodies used were in Fab format. The graph shows response units (RU)as a function of time (sec).

FIG. 12B is a table that summarizes the results of the BIACORE™ SPRanalysis shown in FIG. 12A.

FIG. 13A is a graph showing the results of BIACORE™ SPR analysiscomparing binding of antibody clone h15H6.v2 (parent) and the indicatedvariants at VH position 55 and/or 56 (i.e., HC-D55A HC-G56 (AG), HC-D55EHC-G56 (EG), HC-D55S HC-G56 (SG) and HC-D55 HC-G56A (DA)) to huHtrA1.The antibodies used were in Fab format. The graph shows response units(RU) as a function of time (sec).

FIG. 13B is a table that summarizes the results of the BIACORE™ SPRanalysis shown in FIG. 13A.

FIG. 14A is a graph showing the results of BIACORE™ SPR analysiscomparing binding of antibody clone h15H6.v2 (“parent”) and theindicated combination variants at VL position 94 and VH position 55and/or 56 to huHtrA1. AP_EG: LC-N94A LC-P95 HC-D55E HC-G56 (alsoreferred to as “h15H6.v2.APEG” and “h15H6.v3”). EP_EG: LC-N94E LC-P95HC-D55E HC-G56. QP_EG: LC-N94Q LC-P95 HC-D55E HC-G56. SP_EG: LC-N94SLC-P95 HC-D55E HC-G56.

FIG. 14B is a table that summarizes the results of the BIACORE™ SPRanalysis shown in FIG. 14A.

FIG. 14C is a graph showing the results of a FRET-based blocking assaytesting the ability of h15H6.v2 IgG, h15H6.v2 Fab, and the indicatedcombination variants at VL position 94 and VH position 55 and/or 56 toinhibit the activity of HtrA1. The graph plots percent maximal activityas a function of log antibody concentration (M).

FIG. 14D is a table showing the IC50 values for each of the antibodyclones tested in FIG. 14C.

FIGS. 15A-15B show sequence alignments of the amino acid sequences ofthe VL (FIG. 15A) and VH (FIG. 15B) of anti-HtrA1 antibody clones m19B12and h19B12.v1 compared to the human consensus κ4 sequence (FIG. 15A) orVH3 sequence (FIG. 15B). HVR sequences are delimited by the denotedboxes for each of the antibody clones. The HVR sequences according tothe Kabat definition are underlined. Residues shown in white text inshaded boxes indicates residues that are different between the humanconsensus VH1 sequence and the anti-HtrA1 antibody clones.

FIGS. 16A-16D are graphs showing the results of BIACORE™ SPR analysis ofbinding of antibody clone m19B12 or h19B12.v1 to human or murine HtrA1.Single cycle kinetic analysis was employed. The graphs show responseunits (RU) as a function of time (sec). FIG. 16A shows the results frombinding of m19B12 Fab to huHtrA1. FIG. 16B shows the results frombinding of m19B12 Fab to muHtrA1. FIG. 16C shows the results frombinding of h19B12.v1 Fab to huHtrA1. FIG. 16D shows the results frombinding of h19B12.v1 Fab to huHtrA1. The K_(on), K_(off), and KD foreach analysis are shown as text inside each graph.

FIG. 17 is a schematic diagram outlining the phage panning strategy usedfor NNK deep scanning mutagenesis of the HC and LC HVRs of h15H6.v2 foraffinity maturation.

FIGS. 18A-18B show heatmaps of the log 2 of the enrichment ratio formutations at the indicated VH (FIG. 18A) or VL (FIG. 18B) HVR positionscalculated by dividing the frequency of a given mutation at a givenposition in the sorted sample with the frequency of the very samemutation in the unsorted sample. The enrichment ratios of exemplarymutations are indicated under the heat maps.

FIG. 19 is a table showing mutations identified as being enriched in thesorted sample as compared to the unsorted sample from the NNK librariesand/or soft randomization libraries of the VH and VL of h15v6.v2.

FIG. 20 is a table showing the results of BIACORE™ SPR analysis ofbinding of the indicated affinity matured Fab antibody variant clones.The K_(on), K_(off), and KD for each affinity matured antibody variantclone obtained from this analysis are shown as compared to h15H6.v2 andh15H6.v2.APEG (also referred to as h15H6.v3).

FIGS. 21A-21B show sequence alignments of the amino acid sequences ofthe VL (FIG. 21A) and VH (FIG. 21B) of affinity matured variantanti-HtrA1 antibody clones.

FIG. 22A is a table summarizing the results of the indicated affinitymatured variant anti-HtrA1 Fab antibody clones to inhibit the activityof HtrA1 as assessed in FRET-based H2-Opt activity assays. The tableshows the results from 3 independent experiments as well as the averageand standard deviation (StDev). These experiments employed a rate(RFU/s) analysis.

FIG. 22B is a graph showing an exemplary plot of results from an H2-Optactivity assay using recombinant HtrA1 depicted in FIG. 22A. The assayconditions included 400 pM HtrA1, and 2.5 μM substrate. The buffer was50 mM Tris, 200 mM NaCl, 0.25% CHAPS, pH 8.3. These data are from thesecond repeat of the three independent experiments in FIG. 22A.

FIGS. 23A-23D are graphs showing the results from an H2-Opt activityassay for the indicated h15H6 antibody variant formats analyzed using anRFU/s rate approach. The graphs show percentage of control (RFU/S) as afunction of antibody concentration (nM) for h15H6.v4 IgG monoclonalantibody (mAb) (FIG. 23A), a positive control anti-HtrA1 antibody(YW505.94A IgG, see, e.g., WO 2013/055998) (FIG. 23B), h15H6.v4 Fab(FIG. 23C), and 15H6.v2 Fab (FIG. 23D). A table next to each graph showsthe IC50, Y range, slope factor, and background from each analysis.

FIGS. 23E-23H are graphs showing the results from an H2-Opt activityassay for the indicated h15H6 antibody variant formats analyzed using anendpoint (RFU) approach. The graphs show percentage of control (RFU/S)as a function of antibody concentration (nM) for h15H6.v4 IgG Mab (FIG.23E), a positive control anti-HtrA1 antibody (YW505.94A IgG) (FIG. 23F),h15H6.v4 Fab (FIG. 23G), and 15H6.v2 Fab (FIG. 23H). A table next toeach graph shows the IC50, Y range, slope factor, and background fromeach analysis.

FIGS. 24A-24B are tables showing IC50 (FIG. 24A) and IC90 (FIG. 24B)results for the indicated h15H6 antibody variant formats from a firstset of three independent experiments. The IC50 values were determinedusing 4-parameter fits. The IC90 values were determined from the IC50values and the slopes of the fits. Experiment I corresponds to the datashown in FIGS. 23A-23F. CV %, coefficient of variation. The data wereanalyzed using a rate (RFU/s) approach.

FIGS. 25A-25B are tables showing IC50 (FIG. 25A) and IC90 (FIG. 25B)results for the indicated h15H6 antibody variant formats from a secondset of three independent experiments. The IC50 values were determinedusing 4-parameter fits. The IC90 values were determined from the IC50values and the slopes of the fits. The data were analyzed using either arate (RFU/s) approach or an endpoint (RFU) approach.

FIG. 26A is a graph showing an intact α-casein titration curve. Theexperimental concentration is plotted as a function of theoreticalspike-in concentration (μg/ml). The correlation coefficient (R²) was0.999.

FIG. 26B is a graph showing the results of a mass-spectrometry-basedHtrA1 activity assay. The ability of APEG.LC3.HC3 (h15H6.v4) to inhibitHtra1-PD activity was assessed as described in Example 3, section G. Thesmall molecule inhibitor ucf-101 served as a positive control.

FIGS. 27A-27B are graphs showing the results from two independentendogenous HtrA1 activity assays, Experiment I (FIG. 27A) and ExperimentII (FIG. 27B). For each antibody Fab, #1 and #2 indicate separatedilution series of the same antibody, with initial dilutions performedseparately. The two dilution series were run on the same plate withother reagents being from the same preparation.

FIG. 27C is a table summarizing the results of the endogenous HtrA1activity assays depicted in FIGS. 27A and 27B.

FIG. 28 is a table summarizing the kinetic binding properties andinhibitory activity of the indicated h15H6.v2 Fab variants andderivatives. YW505.94a.28 (see, e.g., WO 2013/055998) served as apositive control. All of the h15H6 Fab variants and derivatives showedimproved affinity and improved potency when compared with YW505.94a.28Fab, with h15H6.v4 Fab showing approximately a 30-fold improvement inaffinity when compared with this antibody.

FIG. 29A shows the amino acid sequence of human HtrA1. The maturesequence is shown in capital letters, the protease domain is underlined,and residues N224 and K248 are shaded.

FIG. 29B shows the amino acid sequence of murine HtrA1. The maturesequence is shown in capital letters, and the protease domain isunderlined.

FIG. 30 shows an alignment of light and heavy chain variable domains ofa reference anti-Factor D antibody (“WT”) and its select variants. HVRswithin the variable domains are underlined. Residue substitutions in thevariants are shown in bold.

FIGS. 31A-31B depict the binding of YW505.94 Fab (as described in WO2013/055998) to HtrA1. When the amino acids designated in FIG. 31A arereplaced with alanine, the binding affinity of the YW505.94 Fab for themutated protein is reduced. The structure shown in FIG. 31B wasgenerated using electron microscopy as described in Ciferri et al.(2015) Biochem. J. 472(2):169-81. The circle shows the epitope for theYW505.94 Fab on the HtrA1 protein. The YW505.94 Fab binds to loops “B”and “C” of the HtrA1 protein.

FIGS. 32A-32B depict the binding of 15H6.v4 Fab to HtrA1, and show thatthe HtrA1 epitope bound by 15H6.v4 Fab is distinct from the epitopebound by YW505.94 Fab. FIG. 32A depicts the interaction between the15H6.v4 Fab and its epitope on the HtrA1 protein, as determined by X-raycrystallography. The 15H6.v4 Fab binds to the LA loop of the HtrA1protein (see, for example, Glaza P et al. (2015) PLoS One10(6):e0131142). The structure shown in FIG. 32B was generated usingelectron microscopy. The circle shows the 15H6V.4 Fab epitope on theHtrA1 protein.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Active” or “activity” or “biological activity” in the context of anantibody of the present invention is the ability to antagonize(partially or fully inhibit) a biological activity of its target, forexample, in vitro and/or in vivo. One example of a biological activityof an antibody is the ability to achieve a measurable improvement in thestate, e.g., pathology, of a disorder associated with its target. Forexample, for an anti-HtrA1 antibody, the disorder may be anHtrA1-associated disorder, such as, for example, AMD (e.g., geographicatrophy). The activity of an anti-HtrA1 antibody can be determined in invitro or in vivo tests, including binding assays, activity assays (e.g.,FRET-based activity assays (e.g., using an H2-Opt substrate) or massspectrometry-based activity assays), using a relevant animal model, orhuman clinical trials. In another example, for an anti-Factor D antibody(e.g., an anti-HtrAlianti-Factor D antibody), the disorder may be acomplement-associated disorder, such as, for example, acomplement-associated ocular disorder. The activity of an anti-Factor Dantibody can be determined in in vitro or in vivo tests, includingbinding assays, alternative pathway hemolysis assays (e.g., assaysmeasuring inhibition of the alternative pathway complement activity oractivation), using a relevant animal model, or human clinical trials.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (KD). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs) and/or frameworkregions (FRs), compared to a parent antibody which does not possess suchalterations, such alterations resulting in an improvement in theaffinity of the antibody for antigen.

The terms “anti-HtrA1 antibody” and “an antibody that specifically bindsto HtrA1” refer to an antibody that is capable of binding HtrA1 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting HtrA1. In one embodiment, theextent of binding of an anti-HtrA1 antibody to an unrelated, non-HtrA1protein is less than about 10% of the binding of the antibody to HtrA1as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to HtrA1 has a dissociation constant (KD) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ Mor less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-HtrA1 antibody binds to an epitope of HtrA1that is conserved among HtrA1 from different species. An anti-HtrA1antibody may be, for example, any anti-HtrA1 antibody described hereinor in International Patent Application Publication No. WO 2013/055998,which is incorporated herein by reference in its entirety.

The terms “anti-Factor D antibody” and “an antibody that specificallybinds to Factor D” refer to an antibody that is capable of bindingFactor D with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting Factor D, for example,in such a manner so as to inhibit or substantially reduce complementactivation. In one embodiment, the extent of binding of an anti-Factor Dantibody to an unrelated, non-Factor D protein is less than about 10% ofthe binding of the antibody to Factor D as measured, e.g., by an RIA. Incertain embodiments, an antibody that binds to Factor D has adissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, ≤0.001 nM, (e.g. 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M,e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-Factor Dantibody binds to an epitope of Factor D that is conserved among FactorD from different species. An anti-Factor D antibody may be anyanti-Factor D antibody described herein and/or in U.S. Pat. Nos.8,067,002; 8,273,352; and 8,268,310; and U.S. patent application Ser.No. 14/700,853, each of which is incorporated herein by reference intheir entirety.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecilic antibodies formed from antibodyfragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire light (L) chain along with the variableregion domain of the heavy (H) chain (VH), and the first constant domainof one heavy chain (CH1). Pepsin treatment of an antibody yields asingle large F(ab′)₂ fragment which roughly corresponds to two disulfidelinked Fab fragments having divalent antigen-binding activity and isstill capable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CH1 domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fe regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, noncovalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeHVRs specific for an antigen) has the ability to recognize and bindantigen, although often at a lower affinity than the entire bindingsite.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, N.Y.,pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodirners of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that contacts an overlapping set of amino acidresidues of the antigen as compared to the reference antibody or blocksbinding of the reference antibody to its antigen in a competition assayby 50% or more. The amino acid residues of an antibody that contact anantigen can be determined, for example, by determining the crystalstructure of the antibody in complex with the antigen or by performinghydrogen/deuterium exchange. In some embodiments, residues of anantibody that are within 5 Å the antigen are considered to contact theantigen. In some embodiments, an antibody that binds to the same epitopeas a reference antibody blocks binding of the reference antibody to itsantigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g., B cell receptor); and Bcell activation.

“Framework” or “framework region” or “FR” refers to variable domainresidues other than hypervariable region (HVR) residues. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3,and FR4.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The “hinge region” is generally defined as stretching from 216-238 (EUnumbering) or 226-251 (Kabat numbering) of human IgG1. The hinge can befurther divided into three distinct regions, the upper, middle (e.g.,core), and lower hinge. In certain embodiments, the hinge region of ahuman IgG1 antibody is generally defined as follows:

The upper hinge comprises amino acids having the sequence EPKSCDKTHT(SEQ ID NO: 157). In certain embodiments, the upper hinge comprises theamino acids at positions 216-225 (EU numbering) or 226-238 (Kabatnumbering).

The middle (e.g., core) hinge comprises amino acids having the sequenceCPPC (SEQ ID NO: 122). In certain embodiments, the core hinge comprisesthe amino acids at positions 226-229 (EU numbering) or 239-242 (Kabatnumbering).

The lower hinge comprises amino acids having the sequence PAPELLGGP (SEQID NO: 158). In certain embodiments, the lower hinge comprises the aminoacids at positions 230-238 (EU numbering) or 243-251 (Kabat numbering).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, FR residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies can comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. Thevariable or “V” domain mediates antigen binding and defines specificityof a particular antibody for its particular antigen. However, thevariability is not evenly distributed across the span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The term “hypervariable region” or“HVR” when used herein refers to the amino acid residues of an antibodywhich are responsible for antigen-binding. The hypervariable regiongenerally comprises amino acid residues from, for example, around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and aroundabout residues 26-35 (H1), 49-65 (H2) and 95-102 (H3) in the VH (in oneembodiment, H1 is around about residues 31-35); Kabat et al., Sequencesof Proteins of Immunological interest, 5th Ed, Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52(L2), and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2), and 96-101(H3) in the VH; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). Thevariable domains of native heavy and light chains each comprise fourFRs, largely adopting a beta-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Accordingly, theHVR and FR sequences generally appear in the following sequence in VH(or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The terms “variable domain residue numbering as in Kabat,” “Kabat aminoacid residue,” or “amino acid position numbering as in Kabat,” andvariations thereof, refers to the numbering system used for heavy chainvariable domains or light chain variable domains of the compilation ofantibodies in Kabat et al., supra. Using this numbering system, theactual linear amino acid sequence may contain fewer or additional aminoacids corresponding to a shortening of, or insertion into, a FR or HVRof the variable domain. For example, a heavy chain variable domain mayinclude a single amino acid insert (residue 52a according to Kabat)after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b,and 82c, etc., according to Kabat) after heavy chain FR residue 82. TheKabat numbering of residues may be determined for a given antibody byalignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term an “isolated antibody” when used to describe the variousantibodies disclosed herein, means an antibody that has been identifiedand separated and/or recovered from a cell or cell culture from which itwas expressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC) approaches. For a review of methods forassessment of antibody purity, see, for example, Flatman et al., J.Chromatogr. B 848:79-87 (2007). In preferred embodiments, the antibodywill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes antibodies in situ withinrecombinant cells, because at least one component of the polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(VH) and a light chain variable domain (VL), where the VH-VL unit haspolyepitopic specificity (i.e., is capable of binding to two differentepitopes on one biological molecule or each epitope on a differentbiological molecule). Such multispecific antibodies include, but are notlimited to, full-length antibodies, antibodies having two or more VL andVH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies,bispecific diabodies and triabodies, antibody fragments that have beenlinked covalently or non-covalently. “Polyepitopic specificity” refersto the ability to specifically bind to two or more different epitopes onthe same or different target(s). “Dual specificity” or “bispecificity”refers to the ability to specifically bind to two different epitopes onthe same or different target(s). However, in contrast to bispecificantibodies, dual-specific antibodies have two antigen-binding arms thatare identical in amino acid sequence and each Fab arm is capable ofrecognizing two antigens. Dual-specificity allows the antibodies tointeract with high affinity with two different antigens as a single Fabor IgG molecule. According to one embodiment, the multispecific antibodyin an IgG1 form binds to each epitope with an affinity of 5 μM to 0.001pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 μM or 0.1 μM to0.001 pM. “Monospecific” refers to the ability to bind only one epitope.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150.000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

With regard to the binding of an antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule. For example, specific binding can be determined by competitionwith a control molecule that is similar to the target, for example, anexcess of non-labeled target. In this case, specific binding isindicated if the binding of the labeled target to a probe iscompetitively inhibited by excess unlabeled target. The term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target as usedherein can be exhibited, for example, by a molecule having a KD for thetarget of 10⁻⁴M or lower, alternatively 10⁻⁵M or lower, alternatively10⁻⁶ M or lower, alternatively 10⁻⁷ M or lower, alternatively 10⁻⁸ M orlower, alternatively 10⁻⁹ M or lower, alternatively 10⁻¹⁰ M or lower,alternatively 10⁻¹¹ M or lower, alternatively 10⁻¹² M or lower or a KDin the range of 10⁻⁴ M to 10⁻⁶ M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹M. As will be appreciated by the skilled artisan, affinity and KD valuesare inversely related. A high affinity for an antigen is measured by alow KD value. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

A “nucleic acid encoding an antibody” refers to one or more nucleic acidmolecules encoding antibody heavy and light chains (or fragmentsthereof), including such nucleic acid molecule(s) in a single vector orseparate vectors, and such nucleic acid molecule(s) present at one ormore locations in a host cell. In some embodiments, the nucleic acidencodes an anti-HtrA1 antibody. In other embodiments, the nucleic acidmay encode an anti-Factor D antibody (e.g., an anti-HtrA1/anti-Factor Dantibody).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

A protein, including an antibody, is said to be “stable” if itessentially retains the intact conformational structure and biologicalactivity. Various analytical techniques for measuring protein stabilityare available in the art and are reviewed in, e.g., Peptide and ProteinDrug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York,N.Y., Pubs. (1991) and Jones (1993) Adv. Drug Delivery Rev. 10: 29-90.An antibody variant with “improved stability” refers to an antibodyvariant that is more stable comparing to the starting referenceantibody. Preferably, antibody variants with improved stability arevariants of the reference (wild-type) antibodies in which specific aminoacid residues are altered for the purpose of improving physicalstability, and/or chemical stability, and/or biological activity, and/orreducing immunogenicity of the native antibodies.

The term “isomerization” refers generally to a chemical process by whicha chemical compound is transformed into any of its isomeric forms, i.e.,forms with the same chemical composition but with different structure orconfiguration and, hence, generally with different physical and chemicalproperties. Specifically used herein is aspartate isomerization, aprocess wherein one or more aspartic acid (D or Asp) residue(s) of apolypeptide have been transformed to isoaspartic acid residue(s). See,e.g., Geiger et al., J. Biol. Chem. 262:785-94, 1987.

The term “deamidation” refers generally to a chemical reaction whereinan amide functional group is removed from an organic compound.Specifically used herein is asparagine deamidation, a process whereinone or more asparagine (N or Asn) residue(s) of a polypeptide (e.g., anantibody) have been converted to aspartic acid (D or Asp), i.e., theneutral amide side chain has been converted to a residue with an overallacidic property. See, e.g., Xie et al., J. Pharm. Sci. 88:8-13, 1999.

An “oxidized” variant of a polypeptide molecule (e.g., an antibody) is apolypeptide wherein one or more methionine (M or Met) or tryptophan (Wor Trp) residue(s) of the original polypeptide have been converted tosulfone or sulfoxide through the sulfur of methionine. Oxidation may beprevented by converting methionine (M or Met) to leucine (L or Leu) orisoleucine (I or Ile). See, e.g., Amphlett et al., Pharm. Biotechnol.,9:1-140, 1996.

Amino acid residues “prone” to certain identified physical or chemicalprocesses (e.g., isomerization, deamidation, or oxidation) refer tothose residues within a specific protein molecule that have beenidentified to have the propensity to undergo the identified processessuch as isomerization, deamidation, or oxidation. Their propensities areoften determined by their relative positions within the primary and/orconformational structure of the protein. For example, it has been shownthat the first Asp in an Asp-XXX motif (wherein XXX can be Asp, Gly,His, Ser or Thr) is prone to Asp isomerization due to the involvement ofits adjacent residue, where some other Asp within the same protein maynot possess such propensity. Assays for identifying residues prone tocertain processes within a specific protein molecule are known in theart. See, e.g., Cacia et al., Biochem. 35:1897-1903, 1996.

The term “HtrA serine peptidase 1 (HtrA1)” or “HtrA1,” as usedinterchangeably herein, refers to any native HtrA1 from any vertebratesource, including mammals such as primates (e.g., humans) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed HtrA1 as well as any form of HtrA1 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of HtrA1, e.g., splice variants or allelic variants.The amino acid sequence of an exemplary human HtrA1 is shown in SEQ IDNO: 121 (see FIG. 29A). The UniProt Accession number for human HtrA1 isQ92743. The amino acid sequence of an exemplary murine HtrA1 is shown inSEQ ID NO: 155 (see FIG. 29B). The UniProt Accession number for murineHtrA1 is Q9R118. As described herein, amino acid residues of huHtrA1 andhuHtrA1 are made with reference to SEQ ID NO: 121 and SEQ ID NO: 155,respectively. Amino acid positions are specified by the one letter aminoacid code followed by its position within SEQ ID NO: 121 or SEQ ID NO:155 (see FIG. 29B). As shown in FIG. 29A, the mature sequence of humanHtrA1 comprises a sequence starting at glutamine at position 23 of SEQID NO: 121 and ending at proline at position 480 of SEQ ID NO:121, e.g.,Q23-P480. Exemplary fragments of human HtrA1 include fragmentscomprising, consisting essentially of, or consisting of amino acidsD161-K379. HtrA1 is also known in the art as protease, serine, 11 (IGFbinding) (PRSS11), ARMD7, HtrA, and IGFBP5-protease.

The term “HtrA1” also encompasses “HtrA1 variants,” which means anactive HtrA1 polypeptide having at least about 80% amino acid sequenceidentity to a native sequence HtrA1 polypeptide, such as SEQ ID NO: 121or SEQ ID NO: 155. Ordinarily, a HtrA1 variant will have at least about80% amino acid sequence identity, or at least about 85% amino acidsequence identity, or at least about 90% amino acid sequence identity,or at least about 95% amino acid sequence identity, or at least about98% amino acid sequence identity, or at least about 99% amino acidsequence identity with a native HtrA1 sequence, e.g., SEQ ID NO: 121 orSEQ ID NO: 155.

The term “HtrA1 binding antagonist” is used in the broadest sense, andincludes any molecule that is capable of neutralizing, blocking,partially or fully inhibiting, abrogating, reducing or interfering withan HtrA1 biological activity. HtrA1 binding antagonists include, withoutlimitation, anti-HtrA1 antibodies, and antibody variants thereof,antigen-binding fragments thereof, other binding polypeptides, peptides,and non-peptide small molecules, that bind to HtrA1 and are capable ofneutralizing, blocking, partially or fully inhibiting, abrogating,reducing or interfering with HtrA1 activities, such as the ability ofHtrA1 to cleave a substrate in vitro (e.g., an H2-Opt substrate orcasein) or in vivo (e.g., the ability of HtrA1 to contribute to thepathology of an ocular disorder (e.g., AMD (e.g., geographic atrophy)).

The term “Factor D,” as used herein, refers to native sequence andvariant Factor D polypeptides. Factor D is also known in the art ascomplement factor D (CFD), C3 proactivator convertase, properdin factorD esterase, and adipsin.

A “native sequence Factor D” is a polypeptide having the same amino acidsequence as a Factor D polypeptide derived from nature, regardless ofits mode of preparation. Thus, native sequence Factor D can be isolatedfrom nature or can be produced by recombinant and/or synthetic means. Inaddition to a mature Factor D protein, such as a mature human Factor Dprotein (see, e.g., NCBI Reference Sequence NM_001928, SEQ ID NO: 126),the term “native sequence Factor D,” specifically encompasses naturallyoccurring precursor forms of Factor D (e.g., an inactive preprotein,which is proteolytically cleaved to produce the active form),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of Factor D, as well asstructural conformational variants of Factor D molecules having the sameamino acid sequence as a Factor D polypeptide derived from nature. TheUniProt Accession Number for human Factor D is P00746. Factor Dpolypeptides of non-human animals, including higher primates andnon-human mammals, are specifically included within this definition.

“Factor D variant” means an active Factor D polypeptide having at leastabout 80% amino acid sequence identity to a native sequence Factor Dpolypeptide, such as the native sequence human Factor D polypeptide(e.g., NM_001928, SEQ ID NO: 126). Ordinarily, a Factor D variant willhave at least about 80% amino acid sequence identity, or at least about85% amino acid sequence identity, or at least about 90% amino acidsequence identity, or at least about 95% amino acid sequence identity,or at least about 98% amino acid sequence identity, or at least about99% amino acid sequence identity with the mature human amino acidsequence (e.g., NM_001928, SEQ ID NO: 126).

The term “Factor D binding antagonist” is used in the broadest sense,and includes any molecule that is capable of neutralizing, blocking,partially or fully inhibiting, abrogating, reducing or interfering witha Factor D biological activity. Factor D binding antagonists include,without limitation, anti-Factor D antibodies, and antibody variantsthereof, antigen-binding fragments thereof, other binding polypeptides,peptides, and non-peptide small molecules, that bind to Factor D and arecapable of neutralizing, blocking, partially or fully inhibiting,abrogating, reducing or interfering with Factor D activities, such asthe ability of Factor D to participate in the pathology of acomplement-associated eye condition.

The term “VEGF antagonist,” as used herein, refers to a molecule capableof binding to VEGF, reducing VEGF expression levels, or neutralizing,blocking, inhibiting, abrogating, reducing, or interfering with VEGFbiological activities, including, but not limited to, VEGF binding toone or more VEGF receptors, VEGF signaling, and VEGF-mediatedangiogenesis and endothelial cell survival or proliferation. Forexample, a molecule capable of neutralizing, blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities canexert its effects by binding to one or more VEGF receptor (VEGFR) (e.g.,VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR)). Included as VEGF antagonists useful inthe methods of the invention are polypeptides that specifically bind toVEGF, anti-VEGF antibodies and antigen-binding fragments thereof,receptor molecules and derivatives which bind specifically to VEGFthereby sequestering its binding to one or more receptors, fusionsproteins (e.g., VEGF-Trap (Regeneron)), and VEGF₁₂₁-gelonin (Peregrine),VEGF antagonists also include antagonist variants of VEGF polypeptides,antisense nucleobase oligomers complementary to at least a fragment of anucleic acid molecule encoding a VEGF polypeptide; small RNAscomplementary to at least a fragment of a nucleic acid molecule encodinga VEGF polypeptide; ribozymes that target VEGF; peptibodies to VEGF; andVEGF aptamers. VEGF antagonists also include polypeptides that bind toVEGFR, anti-VEGFR antibodies, and antigen-binding fragments thereof, andderivatives which bind to VEGFR thereby blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities(e.g., VEGF signaling), or fusions proteins. VEGF antagonists alsoinclude nonpeptide small molecules that bind to VEGF or VEGFR and arecapable of blocking, inhibiting, abrogating, reducing, or interferingwith VEGF biological activities. Thus, the term “VEGF activities”specifically includes VEGF-mediated biological activities of VEGF. Incertain embodiments, the VEGF antagonist reduces or inhibits, by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, theexpression level or biological activity of VEGF. In some embodiments,the VEGF inhibited by the VEGF-specific antagonist is VEGF (8-109), VEGF(1-109), or VEGF₁₆₅.

As used herein, VEGF antagonists can include, but are not limited to,anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab,tanibirumab, aflibercept), anti-VEGFRI antibodies and related molecules(e.g., icrucumab, aflibercept (VEGF Trap-Eye; EYLEA®), andziv-aflibercept (VEGF Trap; ZALTRAP®)), bispecific VEGF antibodies(e.g., MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodiesdisclosed in US 2001/0236388), bispecific antibodies includingcombinations of two of anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms,anti-VEGF antibodies (e.g., bevacizumab, sevacizumab, and ranibizumab),and nonpeptide small molecule VEGF antagonists (e.g., pazopanib,axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib,orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib,apatinib, foretinib, famitinib, and tivozanib).

The terms “anti-VEGF antibody,” an “antibody that binds to VEGF,” and“antibody that specifically binds VEGF” refer to an antibody that iscapable of binding VEGF with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting VEGF. Inone embodiment, the extent of binding of an anti-VEGF antibody to anunrelated, non-VEGF protein is less than about 10% of the binding of theantibody to VEGF as measured, for example, by a radioimmunoassay (RIA).In certain embodiments, an antibody that binds to VEGF has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g., from 10⁻⁸M to 10⁻¹³M,e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments, an anti-VEGFantibody binds to an epitope of VEGF that is conserved among VEGF fromdifferent species.

In certain embodiments, the anti-VEGF antibody can be used as atherapeutic agent in targeting and interfering with diseases orconditions wherein the VEGF activity is involved. Also, the antibody maybe subjected to other biological activity assays, e.g., in order toevaluate its effectiveness as a therapeutic. Such assays are known inthe art and depend on the target antigen and intended use for theantibody. Examples include the HUVEC inhibition assay; tumor cell growthinhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062). An anti-VEGFantibody will usually not bind to other VEGF homologues such as VEGF-Bor VEGF-C, nor other growth factors such as PIGF, PDGF, or bFGF. In oneembodiment, anti-VEGF antibody is a monoclonal antibody that binds tothe same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709. In another embodiment, the anti-VEGF antibodyis a recombinant humanized anti-VEGF monoclonal antibody generatedaccording to Presta et al. (1997) Cancer Res. 57:4593-4599, includingbut not limited to the antibody known as bevacizumab (By; AVASTIN®).

The anti-VEGF antibody “ranibizumab” also known as “LUCENTIS®” or“rhuFab V2” is a humanized, affinity-matured anti-human VEGF Fabfragment. Ranibizumab is produced by standard recombinant technologymethods in Escherichia coli expression vector and bacterialfermentation. Ranibizumab is not glycosylated and has a molecular massof ˜48,000 daltons. See WO 98/45331 and US 2003/0190317. Additionalpreferred antibodies include the G6 or B20 series antibodies (e.g.,G6-31, B20-4.1), as described in PCT Application Publication Nos. WO2005/012359 and WO 2005/044853, which are each incorporated herein byreference in their entirety. For additional preferred antibodies seeU.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332;WO 96/30046; WO94/10202; EP 0666868B1; U.S. Patent ApplicationPublication Nos, 2006009360, 20050186208, 20030206899, 20030190317,20030203409, and 20050112126; and Popkov et al., Journal ofImmunological Methods 288:149-164 (2004). Other preferred antibodiesinclude those that bind to a functional epitope on human VEGF comprisingof residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104 or,alternatively, comprising residues F17, Y21, Q22, Y25, D63, 183, andQ89. Additional anti-VEGF antibodies include anti-VEGF antibodiesdescribed in PCT Application Publication No. WO 2009/155724.

The term “IL-6 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of IL-6 with one or more of its bindingpartners, such as an interleukin-6 receptor (IL-6R) (also called CD126)and/or gp130 (also called CD130). Exemplary IL-6 binding antagonistsinclude, for example, anti-IL-6 antagonists (including anti-IL-6antibodies, e.g., EBI-031 (Eleven Biotherapeutics)) and anti-IL-6Rantagonists (including anti-IL-6R antibodies, e.g., tocilizumab(ACTEMRA®). A “small molecule” is defined herein to have a molecularweight below about 600, preferable below about 1000 daltons.

A “disorder” is any condition that would benefit from treatment with theantibody. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include HtrA1-associated disorders, ocular disorders,and/or complement-associated disorders.

The term “HtrA1-associated disorder,” as used herein, refers in thebroadest sense to any disorder or condition associated with abnormalHtrA1 expression or activities. In some embodiments, HtrA1-associateddisorders are associated with excess HtrA1 levels or activity in whichatypical symptoms may manifest due to the levels or activity of HtrA1locally (e.g., in an eye) and/or systemically in the body. ExemplaryHtrA1-associated disorders include HtrA1-associated ocular disorders,which include, but are not limited to, for example, age-related maculardegeneration (AMD), including wet (exudative) AMD (including early,intermediate, and advanced wet AMD) and dry (nonexudative) AMD(including early, intermediate, and advanced dry AMD (e.g., geographicatrophy (GA)).

As used herein, the term “ocular disorder” includes, but is not limitedto, disorders of the eye including macular degenerative diseases such asage-related macular degeneration (AMD), including wet (exudative) AMD(including early, intermediate, and advanced wet AMD) and dry(nonexudative) AMD (including early, intermediate, and advanced dry AMD(e.g., geographic atrophy (GA)); diabetic retinopathy (DR) and otherischemia-related retinopathies; endophthalmitis; uveitis; choroidalneovascularization (CNV): retinopathy of prematurity (ROP); polypoidalchoroidal vasculopathy (PCV); diabetic macular edema; pathologicalmyopia; von Hippel-Lindau disease; histoplasmosis of the eye; CentralRetinal Vein Occlusion (CRVO); corneal neovascularization; and retinalneovascularization. In some embodiments, the ocular disorder is AMD(e.g., GA).

The term “complement-associated disorder” is used in the broadest senseand includes disorders associated with excessive or uncontrolledcomplement activation. They include complement activation duringcardiopulmonary bypass operations; complement activation due toischemia-reperfusion following acute myocardial infarction, aneurysm,stroke, hemorrhagic shock, crush injury, multiple organ failure,hypobolemic shock, intestinal ischemia, or other events causingischemia. Complement activation has also been shown to be associatedwith inflammatory conditions such as severe burns, endotoxemia, septicshock, adult respiratory distress syndrome, hemodialysis; anaphylacticshock, severe asthma, angioedema, Crohn's disease, sickle cell anemia,poststreptococcal glomerulonephritis, and pancreatitis. The disorder maybe the result of an adverse drug reaction, drug allergy, IL-2 inducedvascular leakage syndrome, or radiographic contrast media allergy. Italso includes autoimmune disease such as systemic lupus erythematosus,myasthenia gravis, rheumatoid arthritis, Alzheimer's disease, andmultiple sclerosis. Complement activation is also associated withtransplant rejection. Complement activation is also associated withocular disorders, such as complement-associated ocular disorders.

The term “complement-associated ocular disorder” is used in the broadestsense and includes all eye conditions the pathology of which involvescomplement, including the classical and the alternative pathways, and inparticular the alternative pathway of complement. Complement-associatedocular disorders include, without limitation, macular degenerativediseases, such as all stages of age-related macular degeneration (AMD),choroidal neovascularization (CNV), uveitis, diabetic and otherischemia-related retinopathies, endophthalmitis, uveitis, and otherintraocular neovascular diseases, such as diabetic macular edema,pathological myopia, von Hippel-Lindau disease, histoplasmosis of theeye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization,and retinal neovascularization. In one example, AMD includes wet AMD(including early, intermediate, and advanced wet AMD) and dry AMD(including early, intermediate, and advanced dry AMD (e.g., geographicatrophy (GA)). In a further example, dry (nonexudative) AMD may includethe presence of hard drusen, soft drusen, geographic atrophy, and/orpigment clumping. Early AMD may include, for example, multiple small toone or more non-extensive medium sized drusen. Intermediate AMD mayinclude, for example, extensive medium drusen to one or more largedrusen. See, e.g., Ferris et al., AREDS Report No. 18; Sallo et al., EyeRes, 34(3):238-40, 2009; Jager et al., New Engl. J. Med., 359(1):1735,2008. In a further example, intermediate dry AMD may include largeconfluent drusen. In a further example, geographic atrophy may includephotoreceptor and/or Retinal Pigmented Epithelial (RPE) loss. In afurther example, the area of geographic atrophy may be small or largeand/or may be in the macula area or in the peripheral retina. In oneexample, the complement-associated ocular disorder is intermediate dryAMD. In one example, complement-associated ocular disorder is geographicatrophy. In one example, the complement-associated ocular disorder iswet AMD (e.g., choroidal neovascularization (CNV)).

The above lists are not all-inclusive, and it will be understood that adisease or disorder may fall within various categories. For example, AMDcan be categorized in some instances as an HtrA1-associated disorder, anocular disorder, and a complement-associated disorder.

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an anti-HtrA1 antibody of the invention, a nucleicacid encoding an anti-HtrA1 antibody of the invention) or a composition(e.g., a pharmaceutical composition, e.g., a pharmaceutical compositionincluding an anti-HtrA1 antibody of the invention) to a subject. Thecompositions utilized in the methods described herein can beadministered, for example, intravitreally (e.g., by intravitrealinjection), ocularly (e.g., by ocular injection), intraocularly (e.g.,by intraocular injection), juxtasclerally (e.g., by juxtascleralinjection), subtenonly (e.g., by subtenon injection), superchoroidally(e.g., by superchoroidal injection), topically (e.g., by eye drop),intramuscularly, intravenously, intradermally, percutaneously,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intrathecally, intranasally, intravaginally, intrarectally,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularly, mucosally, intrapericardially, intraumbilically,intraorbitally, orally, transdermally, by inhalation, by injection, byimplantation, by infusion, by continuous infusion, by localizedperfusion bathing target cells directly, by catheter, by lavage, incremes, or in lipid compositions. The compositions utilized in themethods described herein can also be administered systemically orlocally. The method of administration can vary depending on variousfactors (e.g., the compound or composition being administered and theseverity of the condition, disease, or disorder being treated). Inparticular embodiments, the antibodies described herein (e.g.,anti-HtrA1 antibodies, anti-Factor D antibodies, andanti-HtrA1/anti-Factor D antibodies) are administered by intravitrealinjection.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The terms “long-acting delivery,” “sustained-release,” and “controlledrelease” are used generally to describe a delivery mechanism usingformulation, dosage form, device, or other types of technologies toachieve the prolonged or extended release or bioavailability of atherapeutic agent (e.g., an antibody of the invention). It may refer totechnologies that provide prolonged or extended release orbioavailability of the drug to the general systemic circulation or asubject or to local sites of action in a subject including (but notlimited to) cells, tissues, organs, joints, regions, and the like.Furthermore, these terms may refer to a technology that is used toprolong or extend the release of the drug from a formulation or dosageform, or they may refer to a technology used to extend or prolong thebioavailability or the pharmacokinetics or the duration of action of thedrug to a subject, or they may refer to a technology that is used toextend or prolong the pharmacodynamic effect elicited by a formulation.

A “long-acting formulation,” a “sustained release formulation,” or a“controlled release formulation” is a pharmaceutical formulation, dosageform, or other technology that is used to provide long-acting delivery.In one aspect, the controlled release is used to improve a therapeuticagent's local bioavailability, specifically ocular residence time in thecontext of ocular delivery. “Increased ocular residence time” refers tothe post-delivery period during which the delivered ocular drug remainseffective both in terms of quality (e.g., activity) and in terms ofquantity (e.g., effective amount). In addition to or in lieu of highdose and controlled release, the drug can be modifiedpost-translationally, such as via PEGylation, to achieve increased invivo half-life.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human. A “subject” may be a “patient.”

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of the disease or disorder,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease or disorder, decreasing therate of disease progression, amelioration or palliation of the diseaseor disorder state, and remission or improved prognosis. In someembodiments, antibodies of the invention are used to delay developmentof a disease or disorder or to slow the progression of a disease ordisorder. In some examples, the disorder is an HtrA1-associateddisorder, an ocular disorder, and/or a complement-associated disorder,for example, AMD (e.g., GA).

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the antibody where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells. Theexpression “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (e.g., anti-HtrA1 antibodies of the invention), orthe nucleic acids that encode these sequences, the sequences beingdifferent in the combination of variant amino acids that are introducedinto these sequences, for example, as described herein.

A “mutation” is a deletion, insertion, or substitution of anucleotide(s) relative to a reference nucleotide sequence, such as awild-type sequence.

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art. A codon settypically is represented by 3 capital letters in italics, e.g., NNK,NNS, XYZ, DVK and the like. Synthesis of oligonucleotides with selectednucleotide “degeneracy” at certain positions is well known in that art,for example the TRIM approach (Knappek et al., J. Mol. Biol. 296:57-86,1999; Garrard et al., Gene 128:103, 1993). Such sets of oligonucleotideshaving certain codon sets can be synthesized using commercial nucleicacid synthesizers (available from, for example, Applied Biosystems,Foster City, Calif.), or can be obtained commercially (for example, fromLife Technologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can, but does not necessarily, include restriction enzyme sitesuseful for, for example, cloning purposes.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, for example, filamentous phage, particles. Autility of phage display lies in the fact that large libraries ofrandomized protein variants can be rapidly and efficiently sorted forthose sequences that bind to a target antigen with high affinity.Display of peptide and protein libraries on phage has been used forscreening millions of polypeptides for ones with specific bindingproperties. Polyvalent phage display methods have been used fordisplaying small random peptides and small proteins through fusions toeither gene III or gene VIII of filamentous phage. See, for example,Wells et al., Curr. Opin. Struct. Biol., 3:355-362, 1992, and referencescited therein. In a monovalent phage display, a protein or peptidelibrary is fused to a gene III or a portion thereof, and expressed atlow levels in the presence of wild-type gene III protein so that phageparticles display one copy or none of the fusion proteins. Avidityeffects are reduced relative to polyvalent phage so that sorting is onthe basis of intrinsic ligand affinity, and phagemid vectors are used,which simplify DNA manipulations. See, e.g., Lowman et al., Methods: Acompanion to Methods in Enzymology, 3:205-216, 1991.

A “variant” or “mutant” of a starting or reference polypeptide (e.g., areference antibody or its variable domain(s)/HVR(s)), is a polypeptidethat (1) has an amino acid sequence different from that of the startingor reference polypeptide and (2) was derived from the starting orreference polypeptide through either natural or artificial (man-made)mutagenesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequence of the polypeptide of interest, referred to herein as “aminoacid residue alterations.” Thus, a variant HVR refers to a HVRcomprising a variant sequence with respect to a starting or referencepolypeptide sequence (such as that of a source antibody or antigenbinding fragment). An amino acid residue alteration, in this context,refers to an amino acid different from the amino acid at thecorresponding position in a starting or reference polypeptide sequence(such as that of a reference antibody or fragment thereof). Anycombination of deletion, insertion, and substitution may be made toarrive at the final variant or mutant construct, provided that the finalconstruct possesses the desired functional characteristics. The aminoacid changes also may alter post-translational processes of thepolypeptide, such as changing the number or position of glycosylationsites.

A “wild-type (WI)” or “reference” sequence or the sequence of a“wild-type” or “reference” protein/polypeptide, such as an HVR or avariable domain of a reference antibody, may be the reference sequencefrom which variant polypeptides are derived through the introduction ofmutations. In general, the “wild-type” sequence for a given protein isthe sequence that is most common in nature. Similarly, a “wild-type”gene sequence is the sequence for that gene which is most commonly foundin nature. Mutations may be introduced into a “wild-type” gene (and thusthe protein it encodes) either through natural processes or throughman-induced means. The products of such processes are “variant” or“mutant” forms of the original “wild-type” protein or gene.

A “reference antibody,” as used herein, refers to an antibody orfragment thereof whose antigen-binding sequence serves as the templatesequence upon which diversification according to the criteria describedherein is performed. An antigen-binding sequence generally includes anantibody variable region, preferably at least one HVR, preferablyincluding framework regions.

“Enriched,” as used herein, means that an entity (e.g., an amino acidresidue alteration) is present at a higher frequency in a sorted libraryas compared to a corresponding reference library (e.g., an unsortedlibrary, or a library that has been sorted for a different ornon-relevent antigen). In contrast, “depleted” means that an entity (forexample, an amino acid residue alteration) is present at a lowerfrequency in a sorted library as compared to a corresponding referencelibrary (e.g., an unsorted library, or a library that has been sortedfor a different or non-relevent antigen). The term “neutral,” when usedin reference to methods of identifying amino acid residue variants,means that an entity is neither enriched nor depleted, in other words,it is present at approximately the same frequency in a sorted library ascompared to a corresponding reference library (e.g., an unsortedlibrary, or a library that has been sorted for a different ornon-relevent antigen).

By “isoelectric point (pI)” is meant the pH at which a molecule (e.g., aprotein, such as an antibody) carries no net electrical charge, alsoreferred to in the art as “pH(I)” or “IEP.”

II. Compositions and Methods

The invention provides novel antibodies that bind to HtrA1, and methodsof making and using the same, for example, for therapeutic anddiagnostic uses. Antibodies of the invention are useful, e.g., for thediagnosis or treatment of various disorders, including HtrA1-associateddisorders, ocular disorders, and/or complement-associated disorders,including age-related macular degeneration (e.g., geographic atrophy).

A. Exemplary Anti-HtrA1 Antibodies

In one aspect, the invention is based, in part, on antibodies thatspecifically bind to HtrA1. Antibodies of the invention are useful, forexample, for the treatment or diagnosis of disorders includingHtrA1-associated disorders, ocular disorders, and/orcomplement-associated disorders, including age-related maculardegeneration (e.g., geographic atrophy).

The invention provides isolated antibodies that specifically bind toHtrA1. In some instances, the HtrA1 is human HtrA1 (huHtrA1). In otherinstances, the HtrA1 is murine HtrA1 (muHtrA1). In certain instances, ananti-HtrA1 antibody of the invention specifically binds huHtrA1 with aKD of 100 nM or lower (e.g., 100 nM or lower, 10 nM or lower, 5 nM orlower, 2.5 nM or lower, 1 nM or lower, 100 pM or lower, 10 pM or lower,1 pM or lower, or 0.1 pM or lower). For example, in some instances, ananti-HtrA1 antibody of the invention specifically binds huHtrA1 with aKD of 1 nM or lower (e.g., 1 nM or lower, 900 pM or lower, 800 pM orlower, 700 pM or lower, 600 pM or lower, 550 pM or lower, 500 pM orlower, 400 pM or lower, 300 pM or lower, 200 pM or lower, 150 pM orlower, 125 pM or lower, 100 pM or lower, 75 pM or lower, 50 pM or lower,25 pM or lower, or 1 pM or lower). In some instances, the anti-HtrA1antibody binds huHtrA1 with a KD between about 40 pM and about 700 pM(e.g., between about 40 pM and about 700 pM, between about 40 pM andabout 600 pM, between about 40 pM and about 550 pM, between about 40 pMand about 500 pM, between about 40 pM and about 400 pM, between about 40pM and about 300 pM, between about 40 pM and about 250 pM, between about50 pM and about 200 pM, between about 50 pM and about 175 pM, betweenabout 50 pM and about 150 pM, between about 50 pM and about 125 pM,between about 70 pM and about 125 pM, or between about 50 pM and about125 pM). In some instances, the anti-HtrA1 antibody binds huHtrA1 with aKD of about 110 pM. In some instances, the anti-HtrA1 antibody bindshuHtrA1 with a KD of about 60 pM. Any of the preceding KD values mayrepresent the binding affinity of an anti-HtrA1 antibody of theinvention (e.g., a Fab of an anti-HtrA1 antibody of the invention) tothe protease domain (PD) of huHtrA1 (huHtrA1-PD), for example, asmeasured using BIACORE® surface plasmon resonance, for example, asdescribed herein.

In some instances, an anti-HtrA1 antibody of the invention is capable ofinhibiting the activity of HtrA1. In some instances, the antibodyinhibits the activity of the protease domain of huHtrA1-PD with a 50%inhibitory concentration (IC50) of 10 nM or lower (e.g., 10 nM or lower,5 nM or lower, 2 nM or lower, 1.5 nM or lower, 1 nM or lower, 900 pM orlower, 800 pM or lower, 700 pM or lower, 600 pM or lower, 500 pM orlower, 400 pM or lower, 300 pM or lower, 200 pM or lower, 100 pM orlower, 50 pM or lower, or 1 pM or lower). In some instances, theanti-HtrA1 antibody inhibits the activity of huHtrA1-PD with an IC50 ofbetween about 0.25 nM and about 1 nM (e.g., between about 0.25 nM andabout 1 nM, between about 0.25 nM and about 0.9 nM, between about 0.25nM and about 0.8 nM, between about 0.25 nM and about 0.7 nM, betweenabout 0.25 nM and about 0.6 nM, between about 0.25 nM and about 0.5 nM,or between about 0.25 nM and about 0.4 nM). In some instances, theanti-HtrA1 antibody inhibits the activity of huHtrA1-PD with an IC50 ofabout 0.3 nM. In any of the preceding instances, the inhibitory activitymay be an in vitro FRET-based blocking assay measurement. In someinstances, the in vitro FRET-based blocking assay comprises cleavage ofan H2-Opt probe (e.g., (Mca)IRRVSYSF(Dnp)KK (SEQ ID NO: 152). In any ofthe preceding instances, the IC50 value may be based on the ability ofthe anti-HtrA1 in bivalent format (e.g., IgG format) to inhibithuHtrA1-PD activity.

The invention also encompasses an isolated antibody that specificallybinds to an HtrA1 epitope, where the HtrA1 epitope comprises at leastone amino acid of the HtrA1 protein selected from the group consistingof Arg190, Leu192, Pro193, Phe194, and Arg197, where the amino acidnumbering refers to the numbering for the human HtrA1 precursor protein.In one embodiment, the human HtrA1 precursor protein has the sequence ofSEQ ID NO: 121. In one embodiment, the HtrA1 epitope comprises at leastone amino acid of the HtrA1 protein selected from the group consistingof Leu192, Pro193, and Arg197. In one embodiment, the HtrA1 epitopecomprises at least two amino acids of the HtrA1 protein selected fromthe group consisting of Leu192, Pro193, and Arg197. In a particularembodiment, the HtrA1 epitope comprises the HtrA1 amino acids Leu192,Pro193, and Arg197. In another embodiment, the HtrA1 epitope comprisesthe HtrA1 amino acids Arg190, Leu192. Pro193, and Arg197. In anadditional embodiment, the HtrA1 epitope comprises the HtrA1 amino acidsArg190, Leu192, Pro193, Phe194, and Arg197.

In some embodiments, the anti-HtrA1 antibody when bound to HtrA1 ispositioned 4 angstroms or less from one or more of amino acids Arg190,Leu192, Pro193, Phe194, and Arg197. In some embodiments, the distancebetween the antibody and the one or more amino acids is determined bycrystallography, for example using the crystallography methods describedin the Examples.

In some instances, the anti-HtrA1 antibody may include at least one,two, three, four, five, or six hypervariable regions (HVRs) selectedfrom: (a) HVR-H1 comprising the amino acid sequence of DSEX₁H (SEQ IDNO: 1), wherein X₁ is Met or Leu; (b) HVR-H2 comprising the amino acidsequence of GVDPETX₂GAAYNQKFKG (SEQ ID NO: 2), wherein X₂ is Glu or Asp;(c) HVR-H3 comprising the amino acid sequence of GYDYDYALDY (SEQ ID NO:3); (d) HVR-L1 comprising the amino acid sequence of RASSSVX₃FIH (SEQ IDNO: 4), wherein X₃ is Glu or Asn; (e) HVR-L2 comprising the amino acidsequence of ATSX₄LAS (SEQ ID NO: 5), wherein X₄ is Asn, His or Glu; and(f) HVR-L3 comprising the amino acid sequence of QQWX₅SX₆PWT (SEQ ID NO:6), wherein X₅ is Ser or Tyr and X₆ is Ala or Asn, or a combination ofone or more of the above HVRs and one or more variants thereof having atleast about 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 1-6.

For instance, the anti-HtrA1 antibody may include at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of DSEMH (SEQ ID NO: 7); (b) HVR-H2 comprising theamino acid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) HVR-H3comprising the amino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d)HVR-L1 comprising the amino acid sequence of RASSSVEFIH (SEQ ID NO: 9);(e) HVR-L2 comprising the amino acid sequence of ATSNLAS (SEQ ID NO:10); and (f) HVR-L3 comprising the amino acid sequence of QQWSSAPWT (SEQID NO: 11), ora combination of one or more of the above HVRs and one ormore variants thereof having at least about 80% sequence identity (e.g.,at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs:3 or 7-11.

In some instances, any of the preceding anti-HtrA1 antibodies mayinclude one, two, three, or four of the following heavy chain variabledomain (VH) framework regions (FRs): (a) an FR-H1 comprising the aminoacid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYX₁FX₂ (SEQ ID NO: 12),wherein X₁ is Lys or Thr and X₂ is Thr, Lys, or Arg; (b) an FR-H2comprising the amino acid sequence of WVRQAPGQGLEWIG (SEQ ID NO: 13);(c) an FR-H3 comprising the amino acid sequence ofRATITRDTSTSTAYLELSSLRSEDTAVYYCTR (SEQ ID NO: 14); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

In some instances, any of the preceding anti-HtrA1 antibodies mayinclude one, two, three, or four of the following light chain variabledomain (VL) FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKPLIS (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-HtrA1 antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDSEMH (SEQ ID NO: 7); (b) an HVR-H2 comprising the amino acid sequenceof GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) an HVR-H3 comprising the aminoacid sequence of GYDYDYALDY (SEQ ID NO: 3); (d) an HVR-L1 comprising theamino acid sequence of RASSSVEFIH (SEQ ID NO: 9); (e) an HVR-L2comprising the amino acid sequence of ATSNLAS (SEQ ID NO: 10); and (f)an HVR-L3 comprising the amino acid sequence of QQWSSAPWT (SEQ ID NO:11). In some instances, the anti-HtrA1 antibody includes the followingfour heavy chain variable domain FRs: (a) an FR-H1 comprising the aminoacid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYKFT (SEQ ID NO: 16); (b) anFR-H2 comprising the amino acid sequence of WVRQAPGQGLEWIG (SEQ ID NO:13); (c) an FR-H3 comprising the amino acid sequence ofRATITRDTSTSTAYLELSSLRSEDTAVYYCTR (SEQ ID NO: 14); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15). Infurther instances, the anti-HtrA1 antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKPLIS (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-HtrA1 antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 21 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 22. In some instances,the anti-HtrA1 antibody is APEG.LC3.HC3.

In another example, in some instances, the anti-HtrA1 antibody includesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof DSEMH (SEQ ID NO: 7); (b) an HVR-H2 comprising the amino acidsequence of GVDPETDGAAYNQKFKG (SEQ ID NO: 123); (c) an HVR-H3 comprisingthe amino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASSSVNFIH (SEQ ID NO: 124); (e)an HVR-L2 comprising the amino acid sequence of ATSNLAS (SEQ ID NO: 10);and (f) an HVR-L3 comprising the amino acid sequence of QQWSSAPWT (SEQID NO: 125). In some instances, the anti-HtrA1 antibody includes thefollowing four heavy chain variable domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of QVQLQQSGAELVRPGASVTLSCKASGYTFT (SEQ ID NO:25); (b) an FR-H2 comprising the amino acid sequence of WVKQTPVHGLEWIG(SEQ ID NO: 26); (c) an FR-H3 comprising the amino acid sequence ofKATLTADKSSSTAYMELRSLTSEDSAVYYCTR (SEQ ID NO: 27); and (d) an FR-H4comprising the amino acid sequence of WGQGTSVTVSS (SEQ ID NO: 28). Infurther instances, the anti-HtrA1 antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of NIVVTQSPASLAVSLGQRATISC (SEQ ID NO: 29); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 30);(c) an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 31); and (d) an FR-L4comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 32). Insome instances, the anti-HtrA1 antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 23 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 24. In some instances,the anti-HtrA1 antibody is 15H6 (also referred to as m15H6).

In another example, the anti-HtrA1 antibody may include at least one,two, three, four, five, or six HVRs selected from (a) HVR-H1 comprisingthe amino acid sequence of SYIMS (SEQ ID NO: 39); (b) HVR-H2 comprisingthe amino acid sequence of YISNGGGTTYYSDTIKG (SEQ ID NO: 40); (c) HVR-H3comprising the amino acid sequence of QNFRSDGSSMDY (SEQ ID NO: 41); (d)HVR-L1 comprising the amino acid sequence of RASESVDSYGKSFMH (SEQ ID NO:42); (e) HVR-L2 comprising the amino acid sequence of LASKLES (SEQ IDNO: 43); and (f) HVR-L3 comprising the amino acid sequence of QQNNEDPYT(SEQ ID NO: 44), or a combination of one or more of the above HVRs andone or more variants thereof having at least about 80% sequence identity(e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 39-44.

In some instances, any of the preceding anti-HtrA1 antibodies mayinclude one, two, three, or four of the following heavy chain variabledomain FRs: (a) an FR-H1 comprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 47) orEVKLVESGGGLVEPGGSLKLACVASGFTFS (SEQ ID NO: 57); (b) an FR-H2 comprisingthe amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 48) orWVRQTPEKRLEWVA (SEQ ID NO: 58); (c) an FR-H3 comprising the amino acidsequence of RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 49) orRFTISRDNAKNTLYLQMSTLKSEDTAIYFCAR (SEQ ID NO: 59); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 50) orWGQGTAVTVSS (SEQ ID NO: 60).

In some instances, any of the preceding anti-HtrA1 antibodies mayinclude one, two, three, or four of the following light chain variabledomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 51) or NIVVTQSPASLAVSLGQRATISC (SEQID NO: 61); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGQPPKLLIY (SEQ ID NO: 52) or WYQQKPGQPPKLLIY (SEQ ID NO: 62); (c)an FR-L3 comprising the amino acid sequence ofGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 53) orGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 63); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 54) orFGGGTKLEIK (SEQ ID NO: 64).

For example, in some instances, the anti-HtrA1 antibody includes thefollowing six HVRs: SYIMS (SEQ ID NO: 39); (b) HVR-H2 comprising theamino acid sequence of YISNGGGTTYYSDTIKG (SEQ ID NO: 40); (c) HVR-H3comprising the amino acid sequence of QNFRSDGSSMDY (SEQ ID NO: 41); (d)HVR-L1 comprising the amino acid sequence of RASESVDSYGKSFMH (SEQ ID NO:42); (e) HVR-L2 comprising the amino acid sequence of LASKLES (SEQ IDNO: 43); and (f) HVR-L3 comprising the amino acid sequence of QQNNEDPYT(SEQ ID NO: 44). In some instances, the anti-HtrA1 antibody includes thefollowing four heavy chain variable domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:47); (b) an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWVA(SEQ ID NO: 48); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 49); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 50). Infurther instances, the anti-HtrA1 antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 51); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 52);(c) an FR-L3 comprising the amino acid sequence ofGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 53); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 54). Insome instances, the anti-HtrA1 antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 45 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 46. In some instances,the anti-HtrA1 antibody is h19B12.v1.

In another example, in some instances, the anti-HtrA1 antibody includesthe following six HVRs: SYIMS (SEQ ID NO: 39); (b) HVR-H2 comprising theamino acid sequence of YISNGGGTTYYSDTIKG (SEQ ID NO: 40); (c) HVR-H3comprising the amino acid sequence of QNFRSDGSSMDY (SEQ ID NO: 41); (d)HVR-L1 comprising the amino acid sequence of RASESVDSYGKSFMH (SEQ ID NO:42); (e) HVR-L2 comprising the amino acid sequence of LASKLES (SEQ IDNO: 43); and (f) HVR-L3 comprising the amino acid sequence of QQNNEDPYT(SEQ ID NO: 44). In some instances, the anti-HtrA1 antibody includes thefollowing four heavy chain variable domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of EVKLVESGGGLVEPGGSLKLACVASGFTFS (SEQ ID NO:57); (b) an FR-H2 comprising the amino acid sequence of WVRQTPEKRLEWVA(SEQ ID NO: 58); (c) an FR-H3 comprising the amino acid sequence ofRFTISRDNAKNTLYLQMSTLKSEDTAIYFCAR (SEQ ID NO: 59); and (d) an FR-H4comprising the amino acid sequence of WGQGTAVTVSS (SEQ ID NO: 60). Infurther instances, the anti-HtrA1 antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of NIVVTQSPASLAVSLGQRATISC (SEQ ID NO: 61); (b) an FR-L2comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 62);(c) an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 63); and (d) an FR-L4comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 64). Insome instances, the anti-HtrA1 antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 55 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 56. In some instances,the anti-HtrA1 antibody is 19B12 (also referred to as m19B12).

In another example, an anti-HtrA1 antibody of the invention includesone, two, three, four, five, or six of the HVRs of antibody 20E2 lightand heavy chain variable domains (SEQ ID NOs: 66 and 65, respectively),and wherein (i) the HVR-L1 sequence comprises Kabat amino acid residues24-34, the HVR-L2 sequence comprises Kabat amino acid residues 50-56,and the HVR-L3 sequence comprises Kabat amino acid residues 89-97 of SEQID NO: 66, and (ii) the HVR-H1 sequence comprises Kabat amino acidresidues 31-35, the HVR-H2 sequence comprises Kabat amino acid residues50-65, and the HVR-H3 sequence comprises Kabat amino acid residues95-102 of SEQ ID NO: 65, or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof the above HVRs of antibody 20E2. In some instances, the anti-HtrA1antibody comprises (a) a VH domain comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NOs: 65; (b) a VL domain comprising an amino acidsequence having at least about 90% sequence (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 66; or (c) a VH domain as in (a) and a VL domainas in (b). In some instances, the antibody comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 65 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 66.

In another example, an anti-HtrA1 antibody of the invention includesone, two, three, four, five, or six of the HVRs of antibody 3A5 lightand heavy chain variable domains (SEQ ID NOs: 68 and 67, respectively),and wherein (i) the HVR-L1 sequence comprises Kabat amino acid residues24-34, the HVR-L2 sequence comprises Kabat amino acid residues 50-56,and the HVR-L3 sequence comprises Kabat amino acid residues 89-97 of SEQID NO: 68, and (ii) the HVR-H1 sequence comprises Kabat amino acidresidues 31-35, the HVR-H2 sequence comprises Kabat amino acid residues50-65, and the HVR-H3 sequence comprises Kabat amino acid residues95-102 of SEQ ID NO: 67, or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof the above HVRs of antibody 3A5. In some instances, the anti-HtrA1antibody comprises (a) a VH domain comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NOs: 67; (b) a VL domain comprising an amino acidsequence having at least about 90% sequence (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 68; or (c) a VH domain as in (a) and a VL domainas in (b). In some instances, the antibody comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 67 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 68.

In another example, an anti-HtrA1 antibody of the invention includesone, two, three, four, five, or six of the HVRs of antibody 12A5 lightand heavy chain variable domains (SEQ ID NOs: 70 and 69, respectively),and wherein (i) the HVR-L1 sequence comprises Kabat amino acid residues24-34, the HVR-L2 sequence comprises Kabat amino acid residues 50-56,and the HVR-L3 sequence comprises Kabat amino acid residues 89-97 of SEQID NO: 70, and (ii) the HVR-H1 sequence comprises Kabat amino acidresidues 31-35, the HVR-H2 sequence comprises Kabat amino acid residues50-65, and the HVR-H3 sequence comprises Kabat amino acid residues95-102 of SEQ ID NO: 69, or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof the above HVRs of antibody 12A5. In some instances, the anti-HtrA1antibody comprises (a) a VH domain comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NOs: 69; (b) a VL domain comprising an amino acidsequence having at least about 90% sequence (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 70; or (c) a VH domain as in (a) and a VL domainas in (b). In some instances, the antibody comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 69 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 70.

In some instances, the anti-HtrA1 antibody comprises (a) a VH domaincomprising an amino acid sequence having at least about 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, any one of SEQ ID NOs: 21,23, 76, 77, 78, 93-101, 106, or 107; (b) a VL domain comprising an aminoacid sequence having at least about 90% sequence (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, any one of SEQ ID NOs: 22, 24, 72-74, 81-92, 105, or 108;or (c) a VH domain as in (a) and a VL domain as in (b). For example, insome instances, the antibody comprises a VH domain comprising the aminoacid sequence of SEQ ID NO: 21 and a VL domain comprising the amino acidsequence of SEQ ID NO: 22. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 24. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 76 and a VL domain comprising the amino acidsequence of SEQ ID NO: 72. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 73. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 78 and a VL domain comprising the amino acidsequence of SEQ ID NO: 74. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 87. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 95 and a VL domain comprising the amino acidsequence of SEQ ID NO: 73. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 94 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 73. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 93 and a VL domain comprising the amino acidsequence of SEQ ID NO: 73. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 83. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 97 and a VL domain comprising the amino acidsequence of SEQ ID NO: 73. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 85. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 77 and a VL domain comprising the amino acidsequence of SEQ ID NO: 84. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 100 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 73. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 99 and a VL domain comprising the amino acidsequence of SEQ ID NO: 73. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 101 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 73. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 77 and a VL domain comprising the amino acidsequence of SEQ ID NO: 86. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 96 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 73. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 77 and a VL domain comprising the amino acidsequence of SEQ ID NO: 82. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 88. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 77 and a VL domain comprising the amino acidsequence of SEQ ID NO: 81. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 89. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 98 and a VL domain comprising the amino acidsequence of SEQ ID NO: 73. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 77 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 90. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 95 and a VL domain comprising the amino acidsequence of SEQ ID NO: 83. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 93 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 83. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 94 and a VL domain comprising the amino acidsequence of SEQ ID NO: 87. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 97 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 83. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 94 and a VL domain comprising the amino acidsequence of SEQ ID NO: 83. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 95 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 87. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 94 and a VL domain comprising the amino acidsequence of SEQ ID NO: 85. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 97 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 87. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 93 and a VL domain comprising the amino acidsequence of SEQ ID NO: 87. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 93 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 84. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 97 and a VL domain comprising the amino acidsequence of SEQ ID NO: 85. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 95 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 85. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 93 and a VL domain comprising the amino acidsequence of SEQ ID NO: 85. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 97 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 84. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 95 and a VL domain comprising the amino acidsequence of SEQ ID NO: 84. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 94 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 84. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 104 and a VL domain comprising the amino acidsequence of SEQ ID NO: 22. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 106 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 105. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 107 and a VL domain comprising the amino acidsequence of SEQ ID NO: 105. In some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 106 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 108. In someinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 107 and a VL domain comprising the amino acidsequence of SEQ ID NO: 108.

In some instances, any of the preceding anti-HtrA1 antibodies mayinclude one, two, three, or four of the following heavy chain variabledomain framework regions (FRs): (a) an FR-H1 comprising the amino acidsequence of EVQLVQSGAEVKKPGASVKVSCKASGYX₁FX₂ (SEQ ID NO: 12), wherein X₁is Lys or Thr and X₂ is Thr, Lys, or Arg; (b) an FR-H2 comprising theamino acid sequence of WVRQAPGQGLEWIG (SEQ ID NO: 13); (c) an FR-H3comprising the amino acid sequence of RATITRDTSTSTAYLELSSLRSEDTAVYYCTR(SEQ ID NO: 14); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 15). In other instances, any of the precedinganti-HtrA1 antibody may include one, two, three, or four of thefollowing heavy chain variable domain FRs: (a) an FR-H1 comprising theamino acid sequence of QVQLQQSGAELVRPGASVTLSCKASGYTFT (SEQ ID NO: 25);(b) an FR-H2 comprising the amino acid sequence of WVKQTPVHGLEWIG (SEQID NO: 26); (c) an FR-H3 comprising the amino acid sequence ofKATLTADKSSSTAYMELRSLTSEDSAVYYCTR (SEQ ID NO: 27); and (d) an FR-H4comprising the amino acid sequence of WGQGTSVTVSS (SEQ ID NO: 28).

In some instances, any of the preceding anti-HtrA1 antibodies mayinclude one, two, three, or four of the following light chain variabledomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKPLIS (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20). In other instances, any of the precedinganti-HtrA1 antibody may include one, two, three, or four of thefollowing light chain variable domain FRs: (a) an FR-L1 comprising theamino acid sequence of NIVVTQSPASLAVSLGQRATISC (SEQ ID NO: 29); (b) anFR-L2 comprising the amino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO:30); (c) an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 31); and (d) an FR-L4comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 32).

In some instances, the anti-HtrA1 antibody comprises (a) a VH domaincomprising an amino acid sequence having at least about 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, any one of SEQ ID NOs: 45,55, 65, 67, or 69; (b) a VL domain comprising an amino acid sequencehaving at least about 90% sequence (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of,any one of SEQ ID NOs: 46, 56, 66, 68, or 70; or (c) a VH domain as in(a) and a VL domain as in (b). For example, in some instances, theantibody comprises a VH domain comprising the amino acid sequence of SEQID NO: 45 and a VL domain comprising the amino acid sequence of SEQ IDNO: 46. In some instances, the antibody comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 55 and a VL domain comprising theamino acid sequence of SEQ ID NO: 56. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:65 and a VL domain comprising the amino acid sequence of SEQ ID NO: 66.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 67 and a VL domain comprising theamino acid sequence of SEQ ID NO: 68. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:69 and a VL domain comprising the amino acid sequence of SEQ ID NO: 70.

In some instances, the invention provides an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 21 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 22, such as theantibody referred to herein as APEG.LC3.HC3.

In some instances, the invention provides an isolated antibody thatspecifically binds HtrA1, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 45 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 46, such as theantibody referred to herein as h19B6.v1.

In certain instances, the invention provides an anti-HtrA1 antibody thatbinds to the same epitope as any one of the preceding antibodies. Insome instances, the invention provides an anti-HtrA1 antibody thatcompetes for binding to HtrA1 with any one of the preceding antibodies.

In certain embodiments, any of the preceding anti-HtrA1 antibodies mayhave one or more of the following properties: (i) binds to HtrA1 with aratio of 1 variable domain to one subunit of an HtrA1 trimer (e.g., aFab binds to an HtrA1 trimer with a ratio of 3 Fab to 1 HtrA1 trimer,and an IgG binds to an HtrA1 trimer with a ratio of 3 IgG to 2 HtrA1trimers), (ii) for antibodies comprising two variable domains, binds toHtrA1 in a manner that results in the forming a “cage” similar to thatshown in FIG. 9 of U.S. Patent Application Publication US 2013/0129743,(iii) does not prevent trimer formation of HtrA1, (iv) cross-reacts withmurine HtrA1; (v) does not cross-react with HtrA2, HtrA3 and/or HtrA4;(vi) binds to HtrA1 competitively with anti-HtrA1 antibody YW505.94.28a(see, e.g., WO 2013/055998), (vii) inhibits complex formation betweenHtrA1 and al-antitrypsin (AIAT). In some embodiments, the inventionprovides an antibody that binds to the same epitope as any of thepreceding antibodies.

In a further aspect, an anti-HtrA1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-8 of Section C “AntibodyProperties and Features” below.

B. Exemplary Anti-Factor D Antibodies

The invention provides anti-Factor D antibodies that may be used withthe anti-HtrA1 antibodies of the invention, for example, in methods oftreating a disorder, including an HtrA1-associated disorder, an oculardisorder, and/or a complement-associated disorder (e.g., AMD (e.g.,geographic atrophy)). The invention also provides multispecific (e.g.,bispecific) antibodies that specifically bind to HtrA1 and Factor D(e.g., anti-HtrA1/anti-Factor D antibodies). Any suitable anti-Factor Dantibody may be used in the compositions and methods of the invention.As a non-limiting example, any anti-Factor D antibody described hereinand/or in U.S. Pat. Nos. 8,067,002; 8,268,310; 8,273,352; and/or in U.S.patent application Ser. No. 14/700,853 may be used in the compositionsand methods of the present invention.

For example, in some instances, the anti-Factor D antibody may comprisean amino acid sequence having at least about 80% sequence identity(e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, the monoclonal antibody 166-32 produced from the hybridornadeposited with the American Type Culture Collection (ATCC) anddesignated HB12476. For example, in some instances, the anti-Factor Dantibody comprises (a) a VH domain comprising an amino acid sequencehaving at least about 80% sequence identity (e.g., at least 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:136; (b) a VL domain comprising an amino acid sequence having at leastabout 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, SEQ ID NO: 137; or (c) a VHdomain as in (a) and a VL domain as in (b). For example, in someinstances, the anti-Factor D antibody comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 136 and a VL domain comprising theamino acid sequence of SEQ ID NO: 137, such as the anti-Factor Dmonoclonal antibody 166-32. In some instances, the anti-Factor Dantibody is a humanized derivative of monoclonal antibody 166-32. Insome embodiments, the anti-Factor D antibody binds to the same epitopeas monoclonal antibody 166-32. In some instances, the anti-Factor Dantibody is an antibody fragment derived from monoclonal antibody166-32. In some instances, the antibody fragment derived from monoclonalantibody 166-32 is an Fab, Fab′-SH, Fv, scFv, or an (Fab′)₂ fragment. Insome embodiments, the antibody fragment derived from monoclonal antibody166-32 is an Fab.

In some instances, humanized derivatives of monoclonal antibody 166-32may be employed in the compositions and methods of the invention. Forexample, any humanized derivative of monoclonal antibody 166-32described, for example, in U.S. Pat. No. 8,067,002 may be used in thecompositions and methods of the invention. Exemplary humanizedderivatives of monoclonal antibody 166-32 described in U.S. Pat. No.8,067,002 include, for example, humanized anti-Factor D antibody clones#111, #56, #250, and #416. The amino acid sequences of the VH and VLdomains of humanized anti-Factor D antibody clones #111, #56, #250, and#416 are shown, for example, in FIG. 5 of U.S. Pat. No. 8,067,002. Insome instances, the anti-Factor D antibody may comprise an amino acidsequence having at least about 80% sequence identity (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of,anti-Factor D antibody clone #111, #56, #250, or #416.

In some instances, modified or variant humanized anti-Factor Dantibodies, and fragments thereof, may be used in the compositions andmethods of the invention. For example, any modified or variant versionof humanized anti-Factor D antibody clone #111 described, for example,in U.S. Pat. No. 8,273,352 may be used in the compositions and methodsof the invention, for example, antibody clone 238 or 238-1. In someinstances, the anti-Factor D antibody may comprise an amino acidsequence having at least about 80% sequence identity (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of,anti-Factor D antibody clone 238 or 238-1.

In some instances, the anti-Factor D antibody or antigen-bindingfragment thereof may include at least one, two, three, four, five, orsix HVRs selected from (a) an HVR-H1 comprising the amino acid sequenceof GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2 comprising the amino acidsequence of WINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp orGlu; (c) an HVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ IDNO: 111), wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the aminoacid sequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp orSer, X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprisingthe amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu, or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof SEQ ID NOs: 109-114. For example, in some instances, the anti-FactorD antibody or antigen-binding fragment thereof comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN(SEQ ID NO: 109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp or Glu; (c) anHVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ ID NO: 111),wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the amino acidsequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp or Ser,X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprising theamino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu.

For example, in some instances, the anti-Factor D antibody orantigen-binding fragment thereof may include at least one, two, three,four, five, or six HVRs selected from (a) an HVR-H1 comprising the aminoacid sequence of GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2 comprisingthe amino acid sequence of WINTYTGETTYADDFKG (SEQ ID NO: 115); (c) anHVR-H3 comprising the amino acid sequence of EGGVNN (SEQ ID NO: 116);(d) an HVR-L1 comprising the amino acid sequence of ITSTDIDDDMN (SEQ IDNO: 117); (e) an HVR-L2 comprising the amino acid sequence of GGNTLRP(SEQ ID NO: 113); and (f) an HVR-L3 comprising the amino acid sequenceof LQSDSLPYT (SEQ ID NO: 118), ora combination of one or more of theabove HVRs and one or more variants thereof having at least about 80%sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity)to any one of SEQ ID NOs: 109, 113, or 115-118. For example, in someinstances, the anti-Factor D antibody or antigen-binding fragmentthereof comprises the following six HVRs: (a) an HVR-H1 comprising theamino acid sequence of GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2comprising the amino acid sequence of WINTYTGETTYADDFKG (SEQ ID NO:115); (c) an HVR-H3 comprising the amino acid sequence of EGGVNN (SEQ IDNO: 116); (d) an HVR-L1 comprising the amino acid sequence ofITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2 comprising the amino acidsequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3 comprising theamino acid sequence of LQSDSLPYT (SEQ ID NO: 118).

In some instances, the anti-Factor D antibody comprises (a) a VH domaincomprising an amino acid sequence having at least about 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, any one of SEQ ID NOs: 119,131, or 132; (b) a VL domain comprising an amino acid sequence having atleast about 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of,any one of SEQ ID NOs: 120, 133, 134, or 135; or (c) a VH as in (a) anda VL as in (b). For example, in some instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 119 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 120. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 131 and a VL domain comprising the amino acidsequence of SEQ ID NO: 133. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 131 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 134. In otherinstances, the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO: 131 and a VL domain comprising the amino acidsequence of SEQ ID NO: 135. In other instances, the antibody comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 132 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 135. Any of theanti-Factor D antibodies shown in FIG. 30, or a variant thereof, orafragment thereof, may be used in the compositions and methods of theinvention. In some embodiments, the anti-Factor D antibody orantigen-binding fragment thereof a VH domain comprising the amino acidsequence of SEQ ID NO: 119 and a VL domain comprising the amino acidsequence of SEQ ID NO: 120. In some instances, the anti-Factor Dantigen-binding antibody fragment is lampalizumab having CAS registrynumber 1278466-20-8.

In another example, in some instances, the anti-Factor D antibody is oris derived from a 20D12 antibody, for example, as described in U.S. Pat.No. 8,268,310. In one example, the anti-Factor D antibody includes one,two, three, four, five, or six of the HVRs of antibody 20D12 light andheavy chain variable domains (SEQ ID NOs: 128 and 127, respectively),and wherein (i) the HVR-L1 sequence comprises Kabat amino acid residues24-34, the HVR-L2 sequence comprises Kabat amino acid residues 50-56,and the HVR-L3 sequence comprises Kabat amino acid residues 89-97 of SEQID NO: 128, and (ii) the HVR-H1 sequence comprises Kabat amino acidresidues 31-35, the HVR-H2 sequence comprises Kabat amino acid residues50-65, and the HVR-H3 sequence comprises Kabat amino acid residues95-102 of SEQ ID NO: 127, or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof the above HVRs of antibody 20D12.

In some instances, the anti-Factor D antibody comprises (a) a VH domaincomprising an amino acid sequence having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, SEQ ID NO: 127; (b) a VL domain comprising anamino acid sequence having at least about 80% sequence identity (e.g.,at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 128; or (c) a VH domain as in (a) and a VLdomain as in (b). For example, in some instances, the anti-Factor Dantibody comprises a VH domain comprising the amino acid sequence of SEQID NO: 127 and a VL domain comprising the amino acid sequence of SEQ IDNO: 128, such as the anti-Factor D antibody 20D12. In some instances,the anti-Factor D antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 129 and a light chain comprising the aminoacid sequence of SEQ ID NO: 130. In some instances, the anti-Factor Dantibody is a humanized derivative of monoclonal antibody 20D12. In someembodiments, the anti-Factor D antibody binds to the same epitope asmonoclonal antibody 20D12 or a humanized derivative thereof. In someinstances, the anti-Factor D antibody is an antibody fragment derivedfrom monoclonal antibody 20D12 or a humanized derivative thereof. Insome instances, the antibody fragment derived from monoclonal antibody20D12 or a humanized derivative thereof is an Fab, Fab′-SH, Fv, scFv, oran (Fab′)₂ fragment. In some embodiments, the antibody fragment derivedfrom monoclonal antibody 20D12 or a humanized derivative thereof is anFab.

In some instances, fragments of any of the preceding anti-Factor Dantibodies (e.g., antigen-binding fragments) may be used in thecompositions and methods of the invention. The antibody fragments of thepresent invention may be, for example, Fab, Fab′, F(ab)₂, scFv, (scFv)₂,dAb, hypervariable region (HVR) fragments, linear antibodies,single-chain antibody molecules, minibodies, diabodies, or multispecificantibodies formed from antibody fragments. In a further embodiment, ananti-Factor D antibody fragment (e.g., antigen-binding fragment) that iscapable of penetrating substantially all of the retina may be used inthe compositions and methods of the invention. In an even furtherembodiment, an anti-Factor D antibody fragment (e.g., antigen-bindingfragment) that is capable of penetrating throughout the entire thicknessof the retina may be used in the compositions and methods of theinvention.

In some instances, the invention may include the use of humanizedanti-Factor D antibodies, wherein a Fab fragment of such antibodies havea half life of at least 3, 5, 7, 10, or 12 days after administrationinto a mammalian eye (e.g., human) via a single intravitreal injection.In another embodiment, the invention may include the use of humanizedanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway (AP) complement activation for at least 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 daysafter administration into a mammalian eye (e.g., human) via a singleintravitreal injection. In another embodiment, the invention may includethe use of humanized anti-Factor D antibodies, wherein the concentrationof a Fab fragment of such antibodies that inhibits alternative pathway(AP) complement activation is maintained in retinal tissue for at least40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 days after administration intoa mammalian eye (e.g., human) via a single intravitreal injection. Inanother embodiment, the invention may include the use of humanizedanti-Factor D antibodies, wherein the concentration of a Fab fragment ofsuch antibodies that inhibits alternative pathway (AP) complementactivation is maintained in the vitreous humor for at least 80, 85, 90,95, 100, 105, 110, or 115 days after administration into a mammalian eye(e.g., human) via a single intravitreal injection. In one example, theinvention includes use of a fragment of said anti-Factor D antibodies(e.g., antigen-binding fragments).

In some instances, any of the preceding anti-Factor D antibodies bindsFactor D with a KD of about 20 nM or lower in its monovalent form (e.g.,the KD of the antibody as a Fab fragment to Factor D). In someinstances, an antibody provided herein binds Factor D with a KD of about10 nM or lower in its monovalent form. In some instances, an antibodyprovided herein binds Factor D with a KD of about 5 nM or lower in itsmonovalent form. In some instances, an antibody provided herein bindsFactor D with a KD of about 2 nM or lower in its monovalent Form. Forexample, in some instances, the antibody binds Factor D with a KDbetween about 0.5 pM and about 2 nM (e.g., about 0.5 pM, about 1 pM,about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM,about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25pM, about 50 pM, about 75 pM, about 100 pM, about 125 pM, about 150 pM,about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM,about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400 pM,about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525 pM,about 550 pM, about 575 pM, about 600 pM, about 625 pM, about 650 pM,about 675 pM, about 700 pM, about 725 pM, about 750 pM, about 775 pM,about 800 pM, about 825 pM, about 850 pM, about 875 pM, about 900 pM,about 925 pM, about 950 pM, about 975 pM, about 1 nM, about 1.1 nM,about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM,about 1.7 nM, about 1.8 nM, about 1.9 nM, or about 2 nM) in itsmonovalent form. In some instances, the antibody binds Factor D with aKD between about 0.5 pM and about 100 pM in its monovalent form. In someinstances, the antibody binds Factor D with a KD of about 0.5 pM in itsmonovalent form. In some instances, the antibody binds Factor D with aKD of below about 10 pM in its monovalent form.

In some instances, any of the preceding anti-Factor D antibodies bindsFactor D with a KD of about 10 nM or lower in its bivalent form (e.g.,the KD of the antibody as an IgG to Factor D). In some instances, anantibody provided herein binds Factor D with a KD of about 5 nM or lowerin its bivalent form. In some instances, an antibody provided hereinbinds Factor D with a KD of about 2 nM or lower in its bivalent form.For example, in some instances, the antibody binds Factor D with a KDbetween about 0.5 pM and about 2 nM (e.g., about 0.5 pM, about 1 pM,about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM,about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25pM, about 50 pM, about 75 pM, about 100 pM, about 125 pM, about 150 pM,about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM,about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400 pM,about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525 pM,about 550 pM, about 575 pM, about 600 pM, about 625 pM, about 650 pM,about 675 pM, about 700 pM, about 725 pM, about 750 pM, about 775 pM,about 800 pM, about 825 pM, about 850 pM, about 875 pM, about 900 pM,about 925 pM, about 950 pM, about 975 pM, about 1 nM, about 1.1 nM,about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM,about 1.7 nM, about 1.8 nM, about 1.9 nM, or about 2 nM) in its bivalentForm. In some instances, the antibody binds Factor D with a KD betweenabout 0.5 pM and about 100 pM in its bivalent form. In some instances,the antibody binds Factor D with a KD of about 0.5 pM in its bivalentform. In some instances, the antibody binds Factor D with a KD of belowabout 10 pM in its bivalent form.

In a further aspect, an anti-Factor D antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-8 of Section C “AntibodyProperties and Features” below.

C. Antibody Properties and Features

The antibodies described herein (e.g., anti-HtrA1 antibodies andanti-Factor D antibodies, as described above, as well asanti-HtrA1/anti-Factor D antibodies described below), as well as any ofthe antibodies for use in a method described herein, may have any of thefeatures, singly or in combination, described in Sections 1-8 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein (e.g., an anti-HtrA1antibody, an anti-Factor D antibody, or a bispecific anti-HtrA1anti-Factor D antibody) has a dissociation constant (KD) of ≤1 pM, ≤100nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less,e.g., from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). For example,in some instances, an antibody provided herein binds human HtrA1(huHtrA1) with a KD of about 10 nM or lower. In some instances, anantibody provided herein binds huHtrA1 with a KD of about 5 nM or lower.In some instances, an antibody provided herein binds huHtrA1 with a KDof about 2 nM or lower. For example, in some instances, the antibodybinds huHtrA1 with a KD between about 25 pM and about 2 nM (e.g., about25 pM, about 50 pM, about 75 pM, about 100 pM, about 125 pM, about 150pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275pM, about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400pM, about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525pM, about 550 pM, about 575 pM, about 600 pM, about 625 pM, about 650pM, about 675 pM, about 700 pM, about 725 pM, about 750 pM, about 775pM, about 800 pM, about 825 pM, about 850 pM, about 875 pM, about 900pM, about 925 pM, about 950 pM, about 975 pM, about 1 nM, about 1.1 nM,about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM,about 11 nM, about 1.8 nM, about 1.9 nM, or about 2 nM). In someinstances, the antibody binds huHtrA1 with a KD between about 75 pM andabout 600 pM (e.g., about 75 pM, about 100 pM, about 125 pM, about 150pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275pM, about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400pM, about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525pM, about 550 pM, about 575 pM, about 600 pM). In some instances, theantibody binds huHtrA1 with a KD between about 75 pM and about 500 pM.In some instances, the antibody binds huHtrA1 with a KD between about 75pM and about 400 pM. In some instances, the antibody binds huHtrA1 witha KD between about 75 pM and about 300 pM. In some instances, theantibody binds huHtrA1 with a KD between about 75 pM and about 200 pM.In some instances, the antibody binds huHtrA1 with a KD between about 75pM and about 150 pM. In some instances, the antibody binds huHtrA1 witha KD between about 75 pM and about 125 pM. In some instances, theantibody binds huHtrA1 with a KD between about 75 pM and about 100 pM.In some instances, the antibody binds huHtrA1 with a KD of about 80 pM.In some instances, the antibody binds huHtrA1 with a KD of about 60 pM.In some instances, the antibody binds huHtrA1 with a KD of about 40 pM.

In one embodiment, KD is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, KD is measured using a BIACORE® surfaceplasmon resonance (SPR) assay. For example, an assay using aBIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) isperformed at 25° C. with immobilized antigen CM5 chips at ˜10 responseunits (RU). In one embodiment, carboxymethylated dextran biosensor chips(CM5, BIAcore, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions,Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN®-20) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (KD) is calculated as the ratio k_(off)/k_(on). See, forexample, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette. KD may also be measured usinga BIACORE® SPR assay as described in the Examples below.

2. Antibody Stability

The invention provides antibodies with enhanced stability, for example,as compared to a reference anti-HtrA1 antibody. The stability of anantibody may be determined using any method known in the art, forexample, spectroscopy (e.g., mass spectroscopy), differential scanningfluorimetry (DSF), circular dichroism (CD), intrinsic proteinfluorescence, differential scanning calorimetry, light scattering (e.g.,dynamic light scattering (DLS) and static light scattering (SLS),self-interaction chromatography (SIC). The anti-HtrA1 antibody may have,for example, an enhanced melting temperature (T_(m)), temperature ofaggregation (T_(agg)), or other metrics of stability compared to areference anti-HtrA1 antibody.

The invention provides antibodies with reduced deamidation compared to areference anti-HtrA1 antibody. Deamidation can be reduced or preventedas described herein and/or using methods known in the art. The inventionalso provides antibodies with reduced oxidation (e.g., tryptophanoxidation, for example at position LC-W91), for example, as compared toa reference anti-HtrA1 antibody. Oxidation (e.g., tryptophan oxidation)can be reduced or prevented as described herein and/or using methodsknown in the art. The invention also provides antibodies with reducedisomerization, for example, as compared to a reference anti-HtrA1antibody. Isomerization can be reduced or prevented as described hereinand/or using methods known in the art.

3. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.,Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc.; Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

In some instances, an antibody (e.g., an anti-HtrA1 antibody) providedherein is an Fab. In some embodiments, the Fab comprises a truncation inthe hinge region (e.g., the upper hinge) of the heavy chain constantregion. In some embodiments, the Fab heavy chain constant regionterminates at position 221 (EU numbering). In some embodiments, theamino acid residue at position 221 is an aspartic acid residue (D221).In some embodiments, the heavy chain constant region of the Fabcomprises an amino acid sequence having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, the amino acid sequence of SEQ ID NO: 156. Insome embodiments, the antibody comprises the heavy chain amino acidsequence of SEQ ID NO: 160. In some embodiments, the antibody comprisesthe light chain amino acid sequence of SEQ ID NO: 159. In someembodiments, the antibody comprises the heavy chain amino acid sequenceof SEQ ID NO: 160 and the light chain amino acid sequence of SEQ ID NO:159. In some embodiments, the Fab is an IgG1 Fab.

In some instances, the Fab binds to HtrA1 and may include at least one,two, three, four, five, or six HVRs selected from (a) HVR-H1 comprisingthe amino acid sequence of DSEMH (SEQ ID NO: 7); (b) HVR-H2 comprisingthe amino acid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) HVR-H3comprising the amino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d)HVR-L1 comprising the amino acid sequence of RASSSVEFIH (SEQ ID NO: 9);(e) HVR-L2 comprising the amino acid sequence of ATSNLAS (SEQ ID NO:10); and (f) HVR-L3 comprising the amino acid sequence of QQWSSAPVVT(SEQ ID NO: 11), or a combination of one or more of the above HVRs andone or more variants thereof having at least about 80% sequence identity(e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 3 or 7-11. In some instances, such a Fab may include a truncationin the hinge region (e.g., the upper hinge) of the heavy chain constantregion. In some embodiments, the Fab heavy chain constant regionterminates at position 221 (EU numbering). In some embodiments, theamino acid residue at position 221 is Asp (D221). In some embodiments,the heavy chain constant region of the Fab comprises an amino acidsequence having at least about 80% sequence identity (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of,the amino acid sequence of SEQ ID NO: 156. In some instances, the Fabincludes the following six HVRs: (a) an HVR-H1 comprising the amino acidsequence of DSEMH (SEQ ID NO: 7); (b) an HVR-H2 comprising the aminoacid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) an HVR-H3comprising the amino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d) anHVR-L1 comprising the amino acid sequence of RASSSVEFIH (SEQ ID NO: 9);(e) an HVR-L2 comprising the amino acid sequence of ATSNLAS (SEQ ID NO:10); and (f) an HVR-L3 comprising the amino acid sequence of QQWSSAPWT(SEQ ID NO: 11), and further includes a heavy chain constant regioncomprising the amino acid sequence of SEQ ID NO: 156.

In some instances, the Fab binds to HtrA1 and comprises (a) a VH domaincomprising an amino acid sequence having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, the amino acid sequence of SEQ ID NO: 21; (b) aVL domain comprising an amino acid sequence having at least about 80%sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, the amino acid sequence of SEQ ID NO:22; and (c) a truncation in the hinge region (e.g., the upper hinge) ofthe heavy chain constant region. In some embodiments, the Fab heavychain constant region terminates at position 221 (EU numbering). In someembodiments, the amino acid residue at position 221 is Asp (D221). Insome embodiments, the heavy chain constant region of the Fab comprisesan amino acid sequence having at least about 80% sequence identity(e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity) to, or thesequence of, the amino acid sequence of SEQ ID NO: 156. In someembodiments, the antibody comprises the heavy chain amino acid sequenceof SEQ ID NO: 160. In some embodiments, the antibody comprises the lightchain amino acid sequence of SEQ ID NO: 159. In some embodiments, theantibody comprises the heavy chain amino acid sequence of SEQ ID NO: 160and the light chain amino acid sequence of SEQ ID NO: 159.

In some instances, the Fab binds to HtrA1 and comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 21; (b) a VL domaincomprising the amino acid sequence of SEQ ID NO: 22; and (c) a heavychain constant region comprising the amino acid sequence of SEQ ID NO:156.

4. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, for example, inU.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprisesa non-human variable region (e.g., a variable domain derived from amouse, rat, hamster, rabbit, or non-human primate, such as a monkey) anda human constant domain. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, for example, CDRs, (or portionsthereof) are derived from a non-human antibody, and FRs (or portionsthereof) are derived from human antibody sequences. A humanized antibodyoptionally will also comprise at least a portion of a human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(e.g., the antibody from which the HVR residues are derived), e.g., torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, Front. Biosci, 13:1619-1633 (2008),and are further described, for example, in Riechmann et al., Nature332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321,and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describingspecificity determining region (SDR) grafting); Padlan, Mol. Immunol.28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal., J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol, Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

5. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk et al., Curr.Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol.20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

6. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboorn et al., in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and Wither described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

7. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodiesare monoclonal antibodies that have binding specificities for at leasttwo different sites. In certain embodiments, bispecific antibodies maybind to two different epitopes of HtrA1. In certain embodiments, one ofthe binding specificities is for HtrA1 and the other is for any otherantigen (e.g., a second biological molecule, e.g., Factor D).Accordingly, the bispecific antibody may have binding specificity forHtrA1 and Factor D. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments. Any of the anti-HtrA1 antibodiesdescribed herein may be used to engineer a multispecific antibody (e.g.,a bispecific antibody), for example an anti-HtrA1/anti-Factor Dbispecific antibody. Any of the anti-Factor D antibodies describedherein and/or known in the art may be used to engineer such ananti-HtrA1/anti-Factor D bispecific antibody.

For example, in some instances, a bispecific anti-HtrA1 antibodycomprising a first binding domain that specifically binds HtrA1comprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from: (a) HVR-H1 comprising the amino acidsequence of DSEX₁H (SEQ ID NO: 1), wherein X₁ is Met or Leu; (b) HVR-H2comprising the amino acid sequence of GVDPETX₁GAAYNQKFKG (SEQ ID NO: 2),wherein X₁ is Glu or Asp; (c) HVR-H3 comprising the amino acid sequenceof GYDYDYALDY (SEQ ID NO: 3); (d) HVR-L1 comprising the amino acidsequence of RASSSVX₃FIH (SEQ ID NO: 4), wherein X₃ is Glu or Asn; (e)HVR-L2 comprising the amino acid sequence of ATSX₄LAS (SEQ ID NO: 5),wherein X₄ is Asn, His or Glu; and (f) HVR-L3 comprising the amino acidsequence of QQWX₅SX₆PWT (SEQ ID NO: 6), wherein X₅ is Ser or Tyr and X₆is Ala or Asn, or a combination of one or more of the above HVRs and oneor more variants thereof having at least about 80% sequence identity(e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1-6,may have a second binding domain that binds to Factor D. The secondbinding domain that specifically binds to Factor D may, for example,include at least one, two, three, four, five, or six HVRs selected from(a) an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ IDNO: 109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp or Glu; (c) anHVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ ID NO: 111),wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the amino acidsequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp or Ser,X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprising theamino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu, or a combination of one or more of the aboveHVRs and one or more variants thereof having at least about 80% sequenceidentity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 109-114.

For example, in some instances, a bispecific anti-HtrA1 antibodycomprising a first binding domain that specifically binds HtrA1comprising at least one, two, three, four, five, or six hypervariableregions (HVRs) selected from; (a) HVR-H1 comprising the amino acidsequence of DSEMH (SEQ ID NO: 7); (b) HVR-H2 comprising the amino acidsequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) HVR-H3 comprising theamino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d) HVR-L1 comprisingthe amino acid sequence of RASSSVEFIH (SEQ ID NO: 9); (e) HVR-L2comprising the amino acid sequence of ATSNLAS (SEQ ID NO: 10); and (f)HVR-L3 comprising the amino acid sequence of QQWSSAPWT (SEQ ID NO: 11),or a combination of one or more of the above HVRs and one or morevariants thereof having at least about 80% sequence identity (e.g., 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 3 or 7-11, mayhave a second binding domain that binds to Factor D. The second bindingdomain that specifically binds to Factor D may, for example, include atleast one, two, three, four, five, or six HVRs selected from (a) anHVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ ID NO:109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYADDFKG (SEQ ID NO: 115); (c) an HVR-H3 comprising the aminoacid sequence of EGGVNN (SEQ ID NO: 116); (d) an HVR-L1 comprising theamino acid sequence of ITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2comprising the amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f)an HVR-L3 comprising the amino acid sequence of LQSDSLPYT (SEQ ID NO:118), or a combination of one or more of the above HVRs and one or morevariants thereof having at least about 80% sequence identity (e.g., 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identify) to any one of SEQ ID NOs: 109, 113, or115-118.

In particular embodiments, the invention provides a bispecificanti-HtrA1 antibody that specifically binds both HtrA1 and Factor D,wherein the antibody comprises a first binding domain that specificallybinds HtrA1 comprising the following six HVRs: (a) an HVR-H1 comprisingthe amino acid sequence of DSEMH (SEQ ID NO: 7); (b) an HVR-H2comprising the amino acid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8);(c) an HVR-H3 comprising the amino acid sequence of GYDYDYALDY (SEQ IDNO: 3), (d) an HVR-L1 comprising the amino acid sequence of RASSSVEFIH(SEQ ID NO: 9); (e) an HVR-L2 comprising the amino acid sequence ofATSNLAS (SEQ ID NO: 10); and (f) an HVR-L3 comprising the amino acidsequence of QQWSSAPWT (SEQ ID NO: 11); and a second binding domain thatspecifically binds Factor D comprising the following six HVRs: (a) anHVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ ID NO:109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYADDFKG (SEQ ID NO: 115); (c) an HVR-H3 comprising the aminoacid sequence of EGGVNN (SEQ ID NO: 116); (d) an HVR-L1 comprising theamino acid sequence of ITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2comprising the amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (0an HVR-L3 comprising the amino acid sequence of LQSDSLPYT (SEQ ID NO:118). In some instances, the second binding domain comprises one, two,three, four, five, or six HVRs of the anti-Factor D antigen-bindingantibody fragment lampalizumab.

In some instances, a bispecific anti-HtrA1 antibody comprises a firstbinding domain that specifically binds HtrA1 comprising (a) a VH domaincomprising an amino acid sequence having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, SEQ ID NO: 21; (b) a VL domain comprising anamino acid sequence having at least about 80% sequence identity (e.g.,at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 22; or (c) a VH domain as in (a) and a VL domainas in (b), such as APEG.LC3.HC3, may have a second binding domain thatbinds to Factor D. The second binding domain that specifically binds toFactor D may, for example, comprise (a) a VH domain comprising an aminoacid sequence having at least about 80% sequence identity (e.g., atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 119; (b) a VL domain comprising an amino acid sequencehaving at least about 80% sequence identity (e.g., at least 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:120; or (c) a VH domain as in (a) and a VL domain as in (b). In someinstances, the second binding domain that specifically binds to Factor Dmay comprise (a) a VH domain comprising an amino acid sequence having atleast about 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity) to, or the sequence of, the anti-Factor Dantigen-binding antibody fragment lampalizumab; (b) a VL domaincomprising an amino acid sequence having at least about 80% sequenceidentity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, the anti-Factor D antigen-binding antibodyfragment lampalizumab; or (c) a VH domain as in (a) and a VL domain asin (b).

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see,e.g., Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, for example, in Tutt etal., J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to HtrA1 as well asanother, different antigen (see, e.g., US 2008/0069820).

8. Antibody Variants

In certain embodiments, amino acid sequence variants (e.g., antibodyvariants including one or more amino acid residue alterations) of theantibodies provided herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of an antibodymay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of residues within the amino acid sequences ofthe antibody. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, for example, antigenbinding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Preferred Residue Exemplary Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues and/or FR residues of a parent antibody(e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further study will have modifications (e.g.,improvements) in certain biological properties (e.g., increasedaffinity, increased stability, increased expression, altered pI, and/orreduced immunogenicity) relative to the parent antibody and/or will havesubstantially retained certain biological properties of the parentantibody. An exemplary substitutional variant is an affinity maturedantibody, which may be conveniently generated, for example, using phagedisplay-based affinity maturation techniques such as those describedherein. Briefly, one or more HVR residues are mutated and the variantantibodies displayed on phage and screened for a particular biologicalactivity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues thatcontact antigen, with the resulting variant VH or VL being tested forbinding affinity. Affinity maturation by constructing and reselectingfrom secondary libraries has been described, for example, in Hoogenboomet al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed.,Human Press, Totowa, N.J., (2001)). In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone FOR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. HVR-H3 and HVR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more FRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Suchalterations may, for example, improve antibody affinity and/or stability(e.g., as assessed by an increased melting temperature).

Examples of framework region residues or HVR region residues to modifyinclude possible deamidation sites (i.e., asparagine (N or Asn)),oxidation sites (i.e., methionine (M or Met) or tryptophan (W or Trp))or pyroglutamate conversion sites (i.e., glutamine (Q or Gln)), whereinmodification at such sites prevent or reduce deamidation and/oroxidation and/or pyroglutamate conversion, respectively.

To prevent or reduce the formation of deamidated variants, asparagine (Nor Asn) may be mutated to alanine (A or Ala), glutamine (Q or Gln) orserine (S or Ser). To prevent or reduce the formation of oxidatedvariants, methionine (Met) or tryptophan (W or Trp) may be mutated toleucine (L) or isoleucine (I). To prevent or reduce the formation ofpyroglutamate variants, glutamine (Q or Gln) may be mutated to glutamate(E or Glu). See, e.g., Amphlett et al., Pharm. Biotechnol., 9:1-140,1996. Alternatively, or in addition, one or more alterations (e.g.,substitutions) of framework region residues may be in the Fc region inthe parent antibody.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g., complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, for example, US Patent Publication Nos. US 2003/0157108; US2004/0093621. Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al., J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat ApplNo US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, for example, in WO1997/30087; WO 1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid residue alteration (e.g., asubstitution) at one or more amino acid positions. In certainembodiments, the invention contemplates an antibody variant thatpossesses some but not all effector functions, which make it a desirablecandidate for applications in which the half life of the antibody invivo is important yet certain effector functions (such as complement andADCC) are unnecessary or deleterious. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-492 (1991).

Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362 (see,e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337; and Bruggemann et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc., Mountain View, Calif.; andCYTOTOX 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, for example, in a animal model such as that disclosedin Clynes et al., Proc. Nat'l Acad, Sci. USA 95:652-656 (1998). C1qbinding assays may also be carried out to confirm that the antibody isunable to bind C1q and hence lacks CDC activity. See, for example, C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, e.g.,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg etal., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance/half life determinations canalso be performed using methods known in the art (see, e.g., Petkova etal., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), for example, as described inU.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited towater-soluble polymers. Non-limiting examples of water-soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycolipropylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethyleneimaleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,and the like.

The antibody-polymer conjugates can be made using any suitable techniquefor derivatizing antibody with polymers. It will be appreciated that theinvention is not limited to conjugates utilizing any particular type oflinkage between an antibody or antibody fragment and a polymer.

In one aspect, the conjugates of the invention include species wherein apolymer is covalently attached to a specific site or specific sites onthe parental antibody, i.e., polymer attachment is targeted to aparticular region of a particular amino acid residue or residues in theparental antibody or antibody fragment. Site specific conjugation ofpolymers is most commonly achieved by attachment to cysteine residues inthe parental antibody or antibody fragment. In such embodiments, thecoupling chemistry can, for example, utilize the free sulfhydryl groupof a cysteine residue not in a disulfide bridge in the parentalantibody. The polymer can be activated with any functional group that iscapable of reacting specifically with the free sulihydryl or thiolgroup(s) on the parental antibody, such as maleimide, sulfhydryl, thiol,triflate, tesylate, aziridine, exirane, and 5-pyridyl functional groups.The polymer can be coupled to the parental antibody using any protocolsuitable for the chemistry of the coupling system selected, such as theprotocols and systems described in U.S. Pat. Nos. 4,179,337 and7,122,636; and Jevsevar et al., Biotech, J. 5:113-128, 2010.

In one embodiment, one or more cysteine residue(s) naturally present inthe parental antibody is (are) used as attachment site(s) for polymerconjugation. In another embodiment, one or more cysteine residue(s) is(are) engineered into a selected site or sites in the parental antibodyfor the purpose of providing a specific attachment site or sites forpolymer.

In one aspect, the invention encompasses antibody fragment-polymerconjugates, wherein the antibody fragment is a Fab, and the polymer isattached to one or more cysteine residue in the light or heavy chain ofthe Fab fragment that would ordinarily form the inter-chain disulfidebond linking the light and heavy chains.

In another aspect, the invention encompasses antibody fragment-polymerconjugates, wherein the antibody fragment is a Fab′, and the polymerattachment is targeted to the hinge region of the Fab′ fragment. In oneembodiment, one or more cysteine residue(s) naturally present in thehinge region of the antibody fragment is (are) used to attach thepolymer. In another embodiment, one or more cysteine residues is (are)engineered into the hinge region of the Fab′ fragment for the purpose ofproviding a specific attachment site or sites for polymer. In oneembodiment, a Fab fragment of the invention (e.g., an anti-HtrA1 Fabfragment, an anti-Factor D Fab fragment, or an anti-HtrA1/anti-Factor DFab fragment) is modified by adding one cysteine at the C′-terminal endfor the purpose of providing one attachment site for polymerconjugation. In another embodiment, the Fab fragment of the invention ismodified by adding four additional residues, Cys-Pro-Pro-Cys (SEQ ID NO:122), at the C′-terminal end for the purpose of providing two attachmentsites for polymer conjugation.

One commonly used antibody conjugation is PEGylation, wherein one ormore polyethylene glycol (PEG) polymers are covalently attached to theconstant region of the antibody. See U.S. Pat. Nos. 4,179,337 and7,122,636. PEG polymers of different sizes (e.g., from about 500 D toabout 300,000 D) and shapes (e.g., linear or branched) have been knownand widely used in the field. The polymers useful for the presentinvention may be obtained commercially (e.g., from Nippon Oil and Fats;Nektar Therapeutics; Creative PEGWorks) or prepared fromcommercially-available starting materials using conventional chemicalprocedures. PEGylation changes the physical and chemical properties ofthe antibody drug, and may results in improved pharmacokinetic behaviorssuch as improved stability, decreased immunogenicity, extendedcirculating life, as well as increased residence time. In anotherembodiment, any antibody described herein (e.g., an anti-HtrA1 antibodyof the invention) may be conjugated to hyaluronic acid (HA).

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

f) Isoelectric Point Variants

The invention provides antibodies variants with altered isoelectricpoints. For example, the invention provides antibodies variants with areduced isoelectric point (pI), for example, as compared to a referenceanti-HtrA1 antibody. In some instances, the surface charge is reduced atphysiological pH. In some instances, the anti-HtrA1 antibody has a pIequal to or lower than about 8 (e.g., about 8, about 7, about 6, about5, or about 4). In some instances, the antibody has a pI from about 4 toabout 8 (e.g., about 4, about 5, about 6, about 7, or about 8). In someinstances, the anti-HtrA1 antibody has a pI from about 5 to about 7(e.g., about 5, about 6, or about 7). In some instances, the anti-HtrA1antibody has a pI from about 5 to about 6 (e.g., about 5.1, about 5.2,about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about5.9, or about 6).

Antibodies of the invention may be engineered to have a reduced pI, forexample, by substituting wild-type amino acid residues at a givenposition with an amino acid having a lower pI. The pI of an amino acidcan be determined based on the pKa values of the amine (—NH₂),carboxylic acid (—COOH), and side-chain of the amino acid, which areknown in the art. In some embodiments, surface-exposed amino acidresidues may be substituted to reduce the pI of an antibody. In oneembodiment, surface-exposed amino acid residues may be substituted withglutamate (E). In one embodiment, surface-exposed amino acid residuesmay be substituted with aspartate (D).

D. Recombinant Methods and Compositions

Any of the antibodies (e.g., anti-HtrA1 antibodies) described herein maybe produced using recombinant methods and compositions, for example, asdescribed in U.S. Pat. No. 4,816,567. In one embodiment, an isolatednucleic acid encoding an anti-HtrA1 antibody described herein isprovided. Such a nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such a nucleic acid are provided. In a further embodiment, ahost cell comprising such a nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method ofmaking an anti-HtrA1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-HtrA1 antibody, nucleic acidencoding an antibody, for example, as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, for example, U.S.Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J., 2003), pp. 245-254, describing expression of antibodyfragments in E. coli. After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech, 22:1409-1414 (2004), and Li etal., Nat. Biotech, 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, for example,U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR− CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, for example, Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003).

E. Assays

Anti-HtrA1 antibodies (e.g., anti-HtrA1 antibodies andanti-HtrA1/anti-Factor D antibodies) provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,surface plasmon resonance assays (e.g., BIACORE®), etc.

In one aspect, antigen binding activity (e.g., as indicated by KD) ismeasured using a BIACORE® surface plasmon resonance (SPR) assay. Forexample, an assay using a BIACORE®-2000 or a BIACORE®-3000 (BiAcore,Inc., Piscataway, N.J.) is performed at 25° C. with immobilized antigenCM5 chips at ˜10 response units (RU). In one embodiment,carboxymethylated dextran biosensor chips (CM5, BIAcore, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrarns.The equilibrium dissociation constant (KD) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase of decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette, KD may alsobe measured using a BIACORE® SPR assay as described in the Examplesbelow.

In another aspect, competition assays may be used to identify anantibody that competes with an antibody as described herein for bindingto HtrA1. In certain embodiments, such a competing antibody binds to thesame epitope (e.g., a linear or a conformational epitope) that is boundby an antibody as described herein. Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris(1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized HtrA1 is incubated in asolution comprising a first labeled antibody that binds to HtrA1 and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to HtrA1. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized HtrA1 is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to HtrA1,excess unbound antibody is removed, and the amount of label associatedwith immobilized HtrA1 is measured. If the amount of label associatedwith immobilized HtrA1 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to HtrA1. SeeHarlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-HtrA1 antibodiesthereof having biological activity. Biological activity may include, forexample, inhibiting, blocking, antagonizing, suppressing, interfering,modulating and/or reducing one or more biological activities of HtrA1.Antibodies having such biological activity in vivo and/or in vitro arealso provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. In certain embodiments, an anti-HtrA1 antibodybinds to HtrA1 and reduces or inhibits its serine protease activity forone or more HtrA1 substrates, including, for example, the H2-Optsubstrate, α-casein, β-casein, or BODIPY® FL casein substrates asdescribed in the Examples below, or any other suitable HtrA1 substrate.In certain embodiments, an anti-HtrA1 antibody inhibits HtrA1 serineprotease activity with an IC50 of less than 50 nM, 30 nM, 25 nM, 20 nM,15 nM, 10 nM, 5 nM, 3 nM, 2.5 nM, 2 nM, 1 nM, 800 pM, 600 pM, 500 pM,400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or less for one or more HtrA1substrates. In certain embodiments, an anti-HtrA1 antibody protectsphotoreceptor cells from depredation, protects the thickness of theouter nuclear layer, or protects electroretinogram functional activityin an ocular disease model, such as the constant light exposure mousemodel described in Example 10 of U.S. 2013/0129743.

To determine whether an anti-Factor D antibody, or variant or fragmentthereof (e.g., antigen-binding fragment) is capable of binding to FactorD and exerting a biological effect, for example, inhibition ofalternative pathway hemolysis, hemolytic inhibition assays using rabbitred blood cells (RBCS) may be used, including those described in Example2 of U.S. Pat. No. 8,273,352, which is incorporated herein by referencein its entirety. Such hemolytic inhibition may be determined usingstandard assays (Kostavasili et al., J. Immunology 158:1763-72, 1997;Wiesmann et al., Nature 444:159-60, 2006). Activation of complement insuch assays may be initiated with serum or plasma. Appropriateconcentrations of Factor D in serum or plasma (Pascual et al., KidneyInternational 34:529-536, 1998; Complement Facts Book, Bernard J. Morleyand Mark J. Walport, editors, Academic Press (2000); Barnum et al., J.Immunol. Methods, 67: 303-309, 1984) can be routinely determinedaccording to methods known in the art, including those that have beendescribed in references such as Pascual et al., supra and Barnum et al.,supra, and Example 4 of U.S. Pat. No. 8,273,352. The anti-Factor Dantibodies described herein are generally capable of inhibitingbiological activities associated with Factor D. For example, at aconcentration of 18 μg/ml (equivalent to about 1.5 times the molarconcentration of human factor D in the blood; molar ratio of anti-FactorD antibody to Factor D of about 1.5:1), significant inhibition of thealternative complement activity by the antibody can be observed (see,e.g., U.S. Pat. No. 6,956,107).

3. Stability Assays

In one aspect, assays are provided for determining the stability (e.g.,thermostability) of an anti-HtrA1 antibody. For example, the stabilityof an antibody may be determined using any method known in the art, forexample, differential scanning fluorimetry (DSF), circular dichroism(CD), intrinsic protein fluorescence, differential scanning calorimetry,spectroscopy, light scattering (e.g., dynamic light scattering (DLS) andstatic light scattering (SLS), self-interaction chromatography (SIC).The stability of an assay may be determined as described herein, forexample, using mass spectrometry as described, for example, in Example4, for example in the context of a AAPH stress test and/or a thermalstress test.

F. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-HtrA1 antibodies provided hereinis useful for detecting the presence of HtrA1 in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as a sample comprising photoreceptorcells, retinal pigment epithelium cells, cells of the outer nuclearlayer, the inner nuclear layer, Muller cells, ciliary epithelium, orretinal tissue. In some embodiments, a biological sample comprises abodily fluid, e.g., vitreous or blood.

In one embodiment, an anti-HtrA1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of HtrA1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-HtrA1 antibody as described herein under conditionspermissive for binding of the anti-HtrA1 antibody to HtrA1, anddetecting whether a complex is formed between the anti-HtrA1 antibodyand HtrA1. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-HtrA1 antibody is used to select subjects eligiblefor therapy with an anti-HtrA1 antibody, for example, where HtrA1 is abiomarker for selection of patients.

In certain embodiments, a patient suitable for treatment with ananti-HtrA1 antibody may be identified by detecting one or morepolymorphisms in the HtrA1 gene or HtrA1 control sequence, such as theHtrA1 promoter polymorphism rs11200638(G/A) (see e.g., DeWan et al.,Science 314: 989-992, 2006, which is incorporated herein by reference inits entirety).

Exemplary disorders that may be diagnosed using an antibody of theinvention include, but are not limited to, HtrA-associated disorders,ocular disorders, complement-associated disorders, and preeclampsia. Insome instances, the ocular disorder includes, but is not limited to, forexample, AMD, including wet AMD (including early, intermediate, andadvanced wet AMD) and dry AMD (including early, intermediate, andadvanced dry AMD (e.g., geographic atrophy (GA)), diabetic retinopathy(DR), retinopathy of prematurity (ROP), or polypoidal choroidalvasculopathy (PCV).

In some embodiments, preeclampsia may be diagnosed using an antibody ofthe invention. In some embodiments, an increased level of HtrA1 in asample derived from a subject relative to a reference level of HtrA1 mayindicate that the subject has, or is susceptible to, preeclampsia. See,e.g., Teoh et al. Placenta 36(9):990-995, 2015. In some embodiments,serum HtrA1 levels may be detected using an antibody of the invention.In other embodiments, placental HtrA1 levels may be detected using anantibody of the invention.

In certain embodiments, labeled anti-HtrA1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyze for binding with a limited amountof antibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyze that are boundto the antibodies may conveniently be separated from the standard andanalyze which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, for example, U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

For immunohistochemistry, the sample may be fresh or frozen or may beembedded in paraffin and fixed with a preservative such as formalin, forexample.

G. Diagnostic Kits

As a matter of convenience, an antibody of the present invention (e.g.,an anti-HtrA1 antibody or an anti-HtrA1/anti-Factor D antibody) can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

H. Pharmaceutical Formulations

Therapeutic formulations of the antibody or antibody variant thereof(e.g., an anti-HtrA1 antibody or an anti-HtrA1/anti-Factor D antibody ofthe invention) may be prepared for storage as lyophilized formulationsor aqueous solutions by mixing the polypeptide having the desired degreeof purity with optional “pharmaceutically-acceptable” carriers,excipients, or stabilizers typically employed in the art (all of whichare termed “excipients”). For example, buffering agents, stabilizingagents, preservatives, isotoniliers, non-ionic detergents, antioxidantsand other miscellaneous additives. See e.g., Remington's PharmaceuticalSciences, 16^(th) edition, A. Osol, Ed. (1980). Such additives must benontoxic to the recipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g, fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.), and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, and 3-pentanol.

Isotonicifiers, sometimes known as “stabilizers,” may be added to ensureisotonicity of liquid compositions of the present invention and includepolhydric sugar alcohols, preferably trihydric or higher sugar alcohols,such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (i.e., <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin, or immunoglobulins; hydrophylic polymers, such aspolyvinylpyrrolidone; monosaccharides, such as xylose, mannose,fructose, and glucose; disaccharides such as lactose, maltose, andsucrose; and trisaccacharides such as raffinose; and polysaccharidessuch as dextran. Stabilizers may be present in the range from 0.1 to10.000 weights per part of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic protein (e.g., antibody) aswell as to protect the therapeutic protein against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the protein. Suitablenon-ionic surfactants include polysorbates (20, 80, and the like),polyoxamers (184, 188, and the like), PLURONIC® polyols, polyoxyethylenesorbitan monoethers (TWEEN®-20, TWEEN®-80, and the like). Non-ionicsurfactants may be present in a range of about 0.05 mg/ml to about 1.0mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, and vitamin E), and cosolvents. The formulation hereinmay also contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desireable to include an HtrA1 binding antagonist (e.g., ananti-HtrA1 antibody) and a Factor D binding antagonist (e.g., ananti-Factor D antibody) in the formulation. In another example, fortreating an ocular disorder associated with undesiredneovascularization, such as wet AMD, it may be desirable to furtherprovide an anti-angiogenic therapy, such as a VEGF antagonist therapy,for example, LUCENTIS® (ranibizumab). Such active ingredients aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose, orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin micropheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed, (1980).

Sustained release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, or antibody variant orfragment (e.g., antigen-binding fragment) thereof, which matrices are inthe form of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylenevinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C. resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thiodisulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

I. Therapeutic Methods and Compositions

Any of the anti-HtrA1 antibodies provided herein (e.g., anti-HtrA1antibodies and anti-HtrA1/anti-Factor D antibodies) may be used intherapeutic methods.

In one aspect, an anti-HtrA1 antibody for use as a medicament isprovided. In further aspects, the invention provides an anti-HtrA1antibody for use in treating an HtrA1-associated disorder. In someembodiments, the HtrA1-associated disorder is AMD, including wet AMD(including early, intermediate, and advanced wet AMD) and dry AMD(including early, intermediate, and advanced dry AMD (e.g., geographicatrophy (GA)). In some instances, the AMD is advanced dry AMD (e.g.,GA).

In another embodiment, the invention provides an anti-HtrA1 antibody foruse in treating an ocular disorder. In some instances, the oculardisorder is AMD, including wet (exudative) AMD (including early,intermediate, and advanced wet AMD) and dry (nonexudative) AMD(including early, intermediate, and advanced dry AMD (e.g., GA);diabetic retinopathy (DR) and other ischemic-related retinopathies;endophthalmitis; uveitis; choroidal neovascularization (CNV);retinopathy of prematurity (ROP); polypoidal choroidal vasculopathy(PCV); diabetic macular edema; pathological myopia; von Hippel-Lindaudisease; histoplasmosis of the eye; Central Retinal Vein Occlusion(CRYO); corneal neovascularization; or retinal neovascularization. Insome embodiments, the ocular disorder is AMD (e.g., advanced dry AMD(e.g., GA)).

In another aspect, an anti-HtrA1 antibody for use in a method oftreatment is provided. In certain instances, the invention provides ananti-HtrA1 antibody for use in a method of treating a subject having anHtrA1-associated disorder comprising administering to the individual aneffective amount of the anti-HtrA1 antibody. In some embodiments, theHtrA1-associated disorder is AMD, including wet AMD (including early,intermediate, and advanced wet AMD) and dry AMD (including early,intermediate, and advanced dry AMD (e.g., GA)). In some instances, AMDis advanced dry AMD (e.g., GA).

In another instance, the invention provides an anti-HtrA1 antibody foruse in a method of treating a subject having an ocular disordercomprising administering to the individual an effective amount of theanti-HtrA1 antibody. In some instances, the ocular disorder is AMD,including wet (exudative) AMD (including early, intermediate, andadvanced wet AMD) and dry (nonexudative) AMD (including early,intermediate, and advanced dry AMD (e.g., GA); DR and otherischemia-related retinopathies; endophthalmitis; uveitis; CNV; ROP; PCV;diabetic macular edema; pathological myopia; von Hippel-Lindau disease;histoplasmosis of the eye; CRVO; corneal neovascularization; or retinalneovascularization. In some embodiments, the ocular disorder is AMD(e.g., advanced dry AMD (e.g., GA)).

In some instances, the invention provides an anti-HtrA1 antibody for usein inhibiting retinal or photoreceptor cell degeneration in a subject.In other instances, the invention provides an anti-HtrA1 antibody foruse in inhibiting HtrA1 serine protease activity in an eye of a subject.A “subject” according to any of the above uses may be a human.

The invention provides for the use of an anti-HtrA1 antibody in themanufacture or preparation of a medicament. For example, in oneinstance, the medicament is for treatment of an HtrA1-associateddisorder. In a further instance, the medicament is for use in a methodof treating an HtrA1-associated disorder comprising administering to asubject having an HtrA1-associated disorder an effective amount of themedicament. In any of the preceding uses of medicaments, the method mayinclude administering to the individual an effective amount of at leastone additional therapeutic agent, e.g., as described below. In someembodiments, the HtrA1-associated disorder is AMD, including wet AMD(including early, intermediate, and advanced wet AMD) and dry AMD(including early, intermediate, and advanced dry AMD (e.g., GA)). Insome instances, the AMD is advanced dry AMD (e.g., GA).

In another instance, the medicament is for use in a method of treatingan ocular disorder comprising administering to the subject having anocular disorder an effective amount of the medicament. In someinstances, the ocular disorder is AMD, including wet (exudative) AMD(including early, intermediate, and advanced wet AMD) and dry(nonexudative) AMD (including early, intermediate, and advanced dry AMD(e.g., GA); DR and other ischemia-related retinopathies;endophthalmitis; uveitis; CNV; ROP; PCV; diabetic macular edema;pathological myopia; von Hippel-Lindau disease; histoplasmosis of theeye; CRVO; corneal neovascularization; or retinal neovascularization. Insome embodiments, the ocular disorder is AMD (e.g., advanced dry AMD(e.g., GA)).

The invention provides a method for treating an HtrA1-associateddisorder. In one embodiment, the method comprises administering to asubject having an HtrA1-associated disorder an effective amount of ananti-HtrA1 antibody. In some embodiments, the HtrA1-associated disorderis AMD, including wet AMD (including early, intermediate, and advancedwet AMD) and dry AMD (including early, intermediate, and advanced dryAMD (e.g., GA)). In some instances, the AMD is advanced dry AMD (e.g.,GA). In further instances, the method further comprises administering tothe individual an effective amount of at least one additionaltherapeutic agent, as described below. A “subject” according to any ofthe above methods may be a human.

The invention provides a method for treating an ocular disorder. In oneembodiment, the method comprises administering to a subject having anocular disorder an effective amount of an anti-HtrA1 antibody. In someinstances, the ocular disorder is AMD, including wet (exudative) AMD(including early, intermediate, and advanced wet AMD) and dry(nonexudative) AMD (including early, intermediate, and advanced dry AMD(e.g., GA); DR and other ischemia-related retinopathies;endophthalmitis; uveitis; CNV; ROP; PCV; diabetic macular edema;pathological myopia; von Hippel-Lindau disease; histoplasmosis of theeye; CRVO; corneal neovascularization; or retinal neovascularization. Insome embodiments, the ocular disorder is AMD (e.g., advanced dry AMD(e.g., GA)).

The invention provides a method of treating an HtrA1-associateddisorder, an ocular disorder, and/or a complement-associated disorder ina subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of an HtrA1 bindingantagonist and/or a Factor D binding antagonist. In some embodiments,the HtrA1-associated disorder or complement-associated disorder is anocular disorder. In some embodiments, the ocular disorder is selectedfrom the group consisting of AMD, diabetic retinopathy, choroidalneovascularization (CNV), uveitis, diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, centralretinal vein occlusion, corneal vascularization, and retinalneovascularization. In some instances, the the ocular disorder is AMD,including wet AMD (including early, intermediate, and advanced wet AMD)and dry AMD (including early, intermediate, and advanced dry AMD (e.g.,GA)). In some instances, the AMD is advanced dry AMD (e.g., GA). In anyof the preceding embodiments, the HtrA1-binding antagonist may be ananti-HtrA1 antibody or antigen-binding fragment thereof, for example,any anti-HtrA1 antibody or antigen-binding fragment thereof describedherein. In some embodiments, the antigen-binding antibody fragment isselected from the group consisting of Fab, Fab′-SH, Fv, scFV, and(Fab′)₂ fragments. In some embodiments, the antigen-binding antibodyfragment is an Fab. In some embodiments, the Fab comprises a truncationin the hinge region (e.g., the upper hinge) of the heavy chain constantregion. In some embodiments, the Fab heavy chain constant regionterminates at position 221 (EU numbering). In some embodiments, theamino acid residue at position 221 is an aspartic acid residue. In someembodiments, the heavy chain constant region of the Fab comprises theamino acid sequence of SEQ ID NO: 156. In some embodiments, the Fab isan IgG1 Fab. In some instances, the Factor D binding antagonist is ananti-Factor D antibody or antigen-binding fragment thereof, for example,any of the anti-Factor D antibodies described herein.

In another aspect, the invention provides for the use of a bispecificantibody that specifically binds both HtrA1 and Factor D or anantigen-binding antibody fragment thereof in the manufacture of amedicament for treating a HtrA1-associated disorder, an ocular disorder,and/or a complement-associated disorder. In some embodiments, theHtrA1-associated disorder and/or complement associated disorder is anocular disorder. In some instances, the ocular disorder is AMD,including wet AMD (including early, intermediate, and advanced wet AMD)and dry AMD (including early, intermediate, and advanced dry AMD (e.g.,GA)). In some embodiments, the AMD is advanced dry AMD (e.g., GA). Thebispecific antibody may comprise a binding domain that specificallybinds HtrA1 that is derived from any of the anti-HtrA1 antibodiesdescribed herein. The bispecific antibody may comprise a binding domainthat specifically binds Factor D that is derived from any of theanti-Factor D antibodies described herein. In some embodiments, theantigen-binding antibody fragment is a Fab fragment or an (Fab′)₂fragment.

Any of the anti-Factor D antibodies or antigen-binding fragments thereofdescribed herein and/or known in the art may be used in any of thepreceding methods or uses. For example, in some instances, theanti-Factor D antibody or antigen-binding fragment thereof may includethe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof GYTFTNYGMN (SEQ ID NO: 109); (b) an HVR-H2 comprising the amino acidsequence of WINTYTGETTYAX₁DFKG (SEQ ID NO: 110), wherein X₁ is Asp orGlu; (c) an HVR-H3 comprising the amino acid sequence of EGGVX₁N (SEQ IDNO: 111), wherein X₁ is Asn or Ser; (d) an HVR-L1 comprising the aminoacid sequence of ITSTX₁IX₂X₃DMN (SEQ ID NO: 112), wherein X₁ is Asp orSer, X₂ is Asp or Glu, and X₃ is Asp or Ser; (e) an HVR-L2 comprisingthe amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f) an HVR-L3comprising the amino acid sequence of LQSX₁SLPYT (SEQ ID NO: 114),wherein X₁ is Asp or Glu. In some instances, the anti-Factor D antibodyor antigen-binding fragment thereof includes the following six HVRs: (a)an HVR-H1 comprising the amino acid sequence of GYTFTNYGMN (SEQ ID NO:109); (b) an HVR-H2 comprising the amino acid sequence ofWINTYTGETTYADDFKG (SEQ ID NO: 115); (c) an HVR-H3 comprising the aminoacid sequence of EGGVNN (SEQ ID NO: 116); (d) an HVR-L1 comprising theamino acid sequence of ITSTDIDDDMN (SEQ ID NO: 117); (e) an HVR-L2comprising the amino acid sequence of GGNTLRP (SEQ ID NO: 113); and (f)an HVR-L3 comprising the amino acid sequence of LQSDSLPYT (SEQ ID NO:118). In some embodiments, the anti-Factor D antibody or antigen-bindingfragment thereof includes (a) a VH domain comprising an amino acidsequence having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 119; (b) a VL domain comprising an amino acidsequence having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 120; or (c) a VH domain as in (a) and a VL domainas in (b). In some instances, the VH domain comprises the amino acidsequence of SEQ ID NO: 119. In some instances, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 120. In some instances, theanti-Factor D antigen-binding antibody fragment is lampalizumab havingCAS registry number 1278466-20-8.

It is contemplated that the antibody of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents (e.g., mice and rats) and other mammals inwhich preclinical studies are performed. Such mammals may be establishedanimal models for a disease to be treated with the antibody or may beused to study toxicity of the antibody of interest. In each of theseembodiments, dose escalation studies may be performed in the mammal.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-HtrA1 antibodies provided herein, forexample, for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-HtrA1antibodies provided herein and a pharmaceutically acceptable carrier. Inanother embodiment, a pharmaceutical formulation comprises any of theanti-HtrA1 antibodies provided herein and at least one additionaltherapeutic agent, for example, as described below.

In any of the therapeutic uses and methods described herein, theanti-HtrA1 antibody may be an Fab. In some embodiments, the Fabcomprises a truncation in the hinge region (e.g., the upper hingeregion) of the heavy chain constant region. In some embodiments, the Fabheavy chain constant region terminates at position 221 (EU numbering).In some embodiments, the amino acid residue at position 221 is anaspartic acid residue. In some embodiments, the heavy chain constantregion of the Fab comprises the amino acid sequence of SEQ ID NO: 156.In some embodiments, the Fab is an IgG1 Fab.

An antibody of the invention (and any additional therapeutic agent) forprevention or treatment of an ocular disease or condition can beadministered by any suitable means, including but not limited to, forexample, ocular, intraocular, and/or intravitreal injection, and/orjuxtascleral injection, and/or subtenon injection, and/or superchoroidalinjection, and/or topical administration in the form of eye drops and/orointment. Such antibodies of the invention may be delivered by a varietyof methods, for example, intravitreally as a device and/or a depot thatallows for slow release of the compound into the vitreous, includingthose described in references such as Intraocular Drug Delivery, Jaffe,Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). Inone example, a device may be in the form of a mini pump and/or a matrixand/or a passive diffusion system and/or encapsulated cells that releasethe compound for a prolonged period of time (Intraocular Drug Delivery,Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March2006). Other methods of administration may also be used, which includesbut is not limited to, topical, parenteral, subcutaneous,intraperitoneal, intrapulmonary, intranasal, and intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration.

Formulations for ocular, intraocular, or intravitreal administration canbe prepared by methods and using excipients known in the art. Animportant feature for efficient treatment is proper penetration throughthe eye. Unlike diseases of the front of the eye, where drugs can bedelivered topically, retinal diseases typically benefit from a moresite-specific approach. Eye drops and ointments rarely penetrate theback of the eye, and the blood-ocular barrier hinders penetration ofsystemically administered drugs into ocular tissue. Accordingly, amethod of choice for drug delivery to treat retinal disease, such as AMDand CNV, is typically direct intravitreal injection. Intravitrealinjections are usually repeated at intervals which depend on thepatient's condition, and the properties and half-life of the drugdelivered. For intraocular (e.g., intravitreal) penetration, usuallymolecules of smaller size are preferred.

Eyes have many biophysical and anatomic features that can render oculardrug delivery challenging. For example, blood-ocular barriers aredefense mechanisms for protect the eye from infection, but at the sametime make it hard for drug to penetrate, especially for diseases in theposterior segments of the eye. Consequently, high-dose administration isoften desired to achieve and maintain drug's onsite bioavailability(e.g., ocular residence time) in order to improve efficacy. Meanwhile,the limited space in the back of the eye restrains the drug volume to bedelivered, which in turn may favor drugs to be delivered in a highconcentration formulation.

Patients with ocular disorders (e.g., AMD (e.g., geographic atrophy))can also be benefited from long acting/slow released delivery oftherapeutics. Less frequent dosing would provide improved convenience tothe patient, have potential benefits of decreased infection rate andincreased clinical efficacy. Controlled release of high dose drugs couldalso minimize drug side effects. Two promising systems for long-actingdelivery are PLGA-based solid implants and an implantable port deliverysystem (PDS). Both systems have the potential to provide near zero-orderrelease kinetics for an extended period of time. For PLGA implants, theprotein drug is encapsulated in a hydrophobic polymer matrix and drugrelease is accomplished via slow hydrolysis of the polymer. The rate ofrelease can be controlled by changing the drug loading, polymerhydrophobicity, or polymer molecular weight. The PDS is a refillabledevice where release into the vitreous is controlled by a porous metalmembrane comprising a titanium frit. Since the reservoir has a lowvolume, a high protein concentration is required for effective deliverywith the PDS.

In addition to or in lieu of high concentration and long actingdelivery, increased bioavailability (e.g., ocular residence time) of thedrug can be achieved, or facilitated, by posttranslationalmodifications, wherein the protein drug is covalently conjugated withnatural or synthetic polymers such as polysialylation, HESylation(conjugation with hydroxyethyl starch) and PEGylation. See, e.g., Chenet al., Expert. Opin. Drug Deilv. 8:1221-36, 2011; Konterrnann, BioDrugs23:93-109, 2011. PEGylation, the covalent attachment of polymerpolyethylene glycol (PEG) to a protein, is a well-established technologyespecially useful for extending the half-life of antibody fragmenttherapeutics. Jevsevar et al., Biotech. J. 5:113-128, 2010.

The conditions that a drug is exposed to vary depending on the deliverysystem used. For incorporation into solid PLGA implants, lyophilized orspray-dried drug is used. Implants are produced using a hot-meltextrusion process such that the drug is briefly exposed to temperaturesapproaching 90° C. Although the drug remains in solid state for theduration of release, degradation of PLGA may expose the drug to a low pHenvironment. In contrast, drug delivered with the PDS is maintained athigh concentration in liquid state and exposed to vitreous which ischaracterized as a reducing environment at physiological ionic strengthand pH.

The amount of antibody or antibody variant thereof which will beeffective in the treatment of a particular ocular disorder or conditionwill depend on the nature of the disorder or condition, and can bedetermined by standard clinical techniques. Where possible, it isdesirable to determine the dose-response curve and the pharmaceuticalcompositions of the invention first in vitro, and then in useful animalmodel systems prior to testing in humans.

Additional suitable administration means include parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, forexample, by injections, such as intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein. In some instances, theanti-HtrA1 antibody may be administered intravenously, intramuscularly,intradermally, percutaneously, intraarterially intraperitoneally,intralesionally intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasallyintravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardiallyintraumbilically intraorbitally, orally, topically, transdermally byinhalation, by injection, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions.

The efficacy of the treatment of ocular disorders (e.g.,complement-associated ocular disorders), such as AMD or CNV, can bemeasured by various endpoints commonly used in evaluating intraoculardiseases. For example, vision loss can be assessed. Vision loss can beevaluated by any method known in the art and/or described herein,including but not limited to, for example, measuring by the mean changein best correction visual acuity (BCVA) from baseline to a desired timepoint (e.g., where the BCVA is based on Early Treatment DiabeticRetinopathy Study (ETDRS) visual acuity chart and assessment at a testdistance of 4 meters), measuring the proportion of subjects who losefewer than 15 letters in visual acuity at a desired time point comparedto baseline, measuring the proportion of subjects who gain greater thanor equal to 15 letters in visual acuity at a desired time point comparedto baseline, measuring the proportion of subjects with a visual-acuitySnellen equivalent of 20/2000 or worse at a desired time point,measuring the NEI Visual Functioning Questionnaire, measuring the sizeof CNV and amount of leakage of CNV at a desired time point, e.g., byfluorescein angiography, and the like. Ocular assessments can be done,e.g., which include, but are not limited to, e.g., performing eye exam,measuring intraocular pressure, assessing visual acuity, measuringslitlamp pressure, assessing intraocular inflammation, and the like.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg, 0.2 mg/kg,0.4 mg/kg, 0.6 mg/kg, 0.8 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of antibody canbe an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. In some embodiments, the antibody used is about0.01 mg/kg to about 45 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 1 mg/kg. Onetypical daily dosage might range from about 1 μg/kg to 100 mg/kg ormore, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs.

In some instances, a fixed dose of an anti-HtrA1 antibody of theinvention is administered, for example, to an eye. In some instances,about 0.1 mg to about 10 mg or about 5-15 mg of an anti-HtrA1 antibodyof the invention is administered to an eye, e.g., about 0.1 mg/eye toabout 0.5 mg/eye, about 0.5 mg/eye to about 1 mg/eye, about 1 mg/eye toabout 1.5 mg/eye, about 1.5 mg/eye to about 2 mg/eye, about 2 mg/eye toabout 2.5 mg/eye, about 2.5 mg/eye to about 3 mg/eye, about 3 mg/eye toabout 3.5 mg/eye, about 3.5 mg/eye to about 4 mg/eye, about 4 mg/eye toabout 4.5 mg/eye, about 4.5 mg/eye to about 5 mg/eye, about 5 mg/eye toabout 5.5 mg/eye, about 5.5 mg/eye to about 6 mg/eye, about 6 mg/eye toabout 6.5 mg/eye, about 6.5 mg/eye to about 7 mg/eye, about 7 mg/eye toabout 7.5 mg/eye, about 7.5 mg/eye to about 8 mg/eye, about 8 mg/eye toabout 8.5 mg/eye, about 8.5 mg/eye to about 9 mg/eye, about 9 mg/eye toabout 9.5 mg/eye, or about 9.5 mg/eye to about 10 mg/eye. In someinstances, the antibody is used at about 0.1 mg/eye to about 2 mg/eye,about 0.1 mg/eye to about 3 mg/eye, about 0.1 mg/eye to about 5 mg/eye,about 0.1 mg/eye to about 6 mg/eye, about 0.1 mg/eye to about 7 mg/eye,about 0.1 mg/eye to about 8 mg/eye, about 0.1 mg/eye to about 9 mg/eye,about 0.1 mg/eye to about 10 mg/eye, about 0.5 mg/eye to about 2 mg/eye,about 0.5 mg/eye to about 3 mg/eye, about 1 mg/eye to about 3 mg/eye, orabout 2 mg/eye to about 5 mg/eye. In some instances, a fixed dose of ananti-HtrA1 antibody of about 0.5 mg/eye, about 1 mg/eye, about 1.5mg/eye, about 2 mg/eye, about 2.5 mg/eye, about 3 mg/eye, about 3.5mg/eye, about 4 mg/eye, about 4.5 mg/eye, about 5 mg/eye, about 5.5mg/eye, about 6 mg/eye, about 6.5 mg/eye, about 7 mg/eye, about 7.5mg/eye, about 8 mg/eye, about 8.5 mg/eye, about 9 mg/eye, about 9.5mg/eye, about 10 mg/eye, or more is used. In a particular instance, forexample, a fixed dose of an anti-HtrA1 antibody is administered at about2 mg/eye.

In some embodiments the dose may be administered once a week, once everytwo weeks, once every three weeks, once every four weeks, once everyfive weeks, once every six weeks, once every seven weeks, once everyeight weeks, once every nine weeks, once every ten weeks, once everyeleven weeks, or once every twelve weeks.

An HtrA1 binding antagonist (e.g., an anti-HtrA1 antibody of theinvention) can be administered alone or in combination with at least asecond therapeutic compound. Administration of the HtrA1 bindingantagonist (e.g., an anti-HtrA1 antibody of the invention) and anysecond therapeutic compound can be done simultaneously, e.g., as asingle composition or as two or more distinct compositions using thesame or different administration routes. Alternatively, or additionallythe administration can be done sequentially, in any order. In certainembodiments, intervals ranging from minutes to days, to weeks to months,can be present between the administrations of the two or morecompositions. For example, the HtrA1 binding antagonist (e.g., ananti-HtrA1 antibody of the invention) may be administered first,followed by the second therapeutic compound. However, simultaneousadministration or administration of the second therapeutic compoundprior to the HtrA1 binding antagonist (e.g., an anti-HtrA1 antibody ofthe invention) is also contemplated. In one example, the HtrA1 bindingantagonist is an anti-HtrA1 antibody, for example, any anti-HtrA1antibody described herein or known in the art. In one example, thesecond therapeutic compound is a Factor D binding antagonist. In afurther example, the Factor D binding antagonist is an anti-Factor Dantibody, for example, any anti-Factor D antibody described herein orknown in the art. In particular embodiments, the anti-Factor D antibodyis lampalizumab. In further embodiments, the anti-Factor D antibody isadministered at a close of 1-15 mgs, for example at a close of 10 mgs.In particular embodiments, the lampalizumab is administered once everytwo weeks, once every three weeks, or once every four weeks. In certainembodiments, an additional therapeutic agent is a therapeutic agentsuitable for treatment of an ocular disorder associated with undesirableneovascularization in the eye, such as, for example, wet AMD. Suitabletherapeutic agents include, for example, anti-angiogenic therapies suchas VEGF antagonists (e.g., anti-VEGF antibodies and antibody fragments,including LUCENTIS® (ranibizumab), and anti-VEGFR1 antibodies andrelated molecules (e.g., aflibercept (VEGF Trap-Eye; EYLEA®));inhibitors of the complement system, such as complement factor C2antagonists (including, for example, anti-CFC2 antibodies); andanti-inflammatory agents, such as IL-6 binding antagonists (e.g.,tocilizurnab (ACTEMRA®) and EBI-031 (Eleven Biotherapeutics)). In otherembodiments, treatment of a disease or disorder associated withundesirable ocular neovascularization may involve a combination of ananti-HtrA1 antibody and photodynamic therapy (e.g., with MACUGEN™ orVISUDYNE™).

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theHtrA1 binding antagonist (e.g., anti-HtrA1 antibody) and administrationof an additional therapeutic agent occur within about one month, orwithin about one, two or three weeks, or within about one, two, three,four, five, or six days, of each other.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-HtrA1 antibody.

J. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Development of Stable, High-Potency, High-Affinity Antibodies that BindHtrA1

The goal of the following experiments was to discover new anti-HtrA1antibodies having higher potency and higher affinity for HtrA1.

First, mHtrA1 knock-out mice were generated. It was necessary to useknock-out mice because initial efforts to generate hybridomas from miceexpressing HtrA1 were not successful, likely because the murine andhuman HtrA1 proteins share 98% sequence identify.

Next, the HtrA1 knock-out mice were immunized with the protease domainof mHtrA1. The resulting hybridomas were screened by ELISA and 75 ELISApositive clones were identified. The 75 clones were tested for theability to inhibit cleavage of an HtrA1 protease substrate. 10 of the 75clones were shown to inhibit HtrA1 protease activity.

Seven of these clones were selected for further analysis based on theirability to inhibit protease activity, to bind human and murine HtrA1,and to selectively bind muHtrA1 over muHtrA3 and muHtrA4. These sevenclones underwent further screening and four were found to have improvedpotency for HtrA1 when compared with the control antibody YW505.94. Twoof these antibodies, 15H6 and 9B12, were selected for furtherdevelopment based on their preferred molecular profile, includingpotency and selectivity.

The hypervariable regions of the selected antibodies were grafted onto ahuman framework as outlined below. The importance of the mouse lightchain Vernier references was tested for 15H6 by individually swappingthese residues and testing binding to human and mouse HtrA1. Onesubstitution abolished binding, two reduced binding, two affected onlybinding to murine HtrA1, and three did not have a significant impact onbinding. These three substitutions were introduced into antibody 15H6.v1to make antibody 15H6.v2

15H6.v2 was then was engineered to improve its stability. Potentiallyunstable residues that could lead to oxidation (W91 in HVR-L3), clipping(N94 P95 in HVR-LC), and deamidation (D55 G56 in HVR-H2) wereidentified. Substitutions of W91 substantially impacted binding. Foursubstitutions at position N94 were tested. Two of these substitutionsimpacted binding, but two were selected for additional analysis. Foursubstitutions at position D55 were tested. Only one of thesesubstitutions showed comparable binding to the parent molecule.

The next step was to improve the affinity of h15H6.v2 binding to HtrA1.As a first step, deep sequencing was used to investigate thestructure/function relationship for h15H6.v2. Of the many individualmutations tested in this experiment, approximately 19 individualmutations were found to improve affinity for HtrA1. Antibodiescontaining combinations the LC and HC mutations with the slowestoff-rate were designed and tested, resulting in the identification ofvariant antibodies having improved potency and affinity for HtrA1.Variants having the best affinity and potency were also engineered tointroduce the stabilizing substitutions described above. Of theresulting variants, h15H6.v4 was found to have the highest potencyagainst HtrA1.

The structure of h15H6.v4 in Fab format bound to HtrA1 was determined byX-ray crystallography and electron microscopy. This structural analysisrevealed that the h15H6.v4 Fab binds closely to the “LA loop” of theHtrA1 protein. The epitope bound by h15H6.v4 is distinct from that boundby control antibody YW505.94. Although it is not intended that thepresent invention be bound by any particular mechanism, it is possiblethat the close interaction between h15H6.v4 and the LA loop of HtrA1explains the significantly improved affinity and potency of thisantibody when compared with YW505.94.

The experiments described above are outlined in greater detail below.

Example 1: Generation of Anti-HtrA1 Antibodies Using HybridomaApproaches

A. Media and Antibodies

CLONACELL™-HY Medium B (Cat #03802), Medium C (Cat #03803), Medium D(Cat #03804) and Medium E (Cat #03805) were from StemCell Technologies.CYTOFUSION® Medium C (Cat #LCM-C) used for electrofusion was from CytoPulse Sciences. Allophycocyanin (APC)-labeled goat F(ab′)₂ anti-mouse Igwas from SouthernBiotech (Cat #1012-11), horseradish peroxidase(HRP)-conjugated goat anti-mouse IgG Fc antibody was from Sigma. TMB(3,3′,5,5′-tetramethylbenzidine) One Component HRP Microwell Substrate(Cat #TMBW-1000-01) and TMB Stop Reagent (Cat #BSTP-1000-01) were fromBioFx Laboratories.

B. In Vivo Immunization of Mice

Five HtrA1-knockout mice (HtrA1.noneo.BALB.ko.C1-3) were immunized withpurified His-tagged recombinant murine HtrA1 protease domain (referredto herein as muHtrA1-PD-His, SEQ ID NO: 153; see Example 2 of WO2013/055998) suspended in monophosphoryl lipid Aitrehalosedicorynomycolate adjuvant by footpad injection (2 μg/injection permouse) at 3 to 4 day intervals for a total of 12 boosts, followed by 2pre-fusion boosts with antigen in phosphate-buffered saline (PBS). Threedays after the final boost, lymphocytes from immunized mice spleens andlymph nodes were harvested and isolated. muHtrA1-PD-His was prepared forinjection by purification as described in Example 2 of WO 2013/055998.

C. Cell Fusion, Hybridoma Screening, and Subcloning

Isolated mouse spleen cells from two mice (numbers 748 and 749) werefused with SP2/0 myeloma cells (American Type Culture Collection) usingthe Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences). Briefly, afterwashing twice with CYTOFUSION® Medium C, the isolated spleen cells andSP2/0 cells were mixed at a 1:1 ratio and then resuspended inCYTOFUSION® Medium C at a concentration of 10 million cells/ml.Electrofusion was performed according to the manufacturer's guidance.Fused cells were cultured in CLONACELL™-HY Medium C overnight at 37° C.in a 7% CO₂ incubator. The next day, the fused cells were centrifugedand then resuspended in 10 ml CLONACELL™-HY Medium C and then gentlymixed with 90 ml methylcellulose-based CLONACELL™-HY Medium D containingthe selective reagents hypoxanthine, aminopterin and thymidine (HAT).The cells were plated into 40×100 mm Petri dishes (Cat #351029, BectonDickinson) and allowed to grow in 37° C. in a 7% CO₂ incubator. After 10days incubation, 1429 single hybridoma clones were picked using aCLONEPIX™ system (Genetix, United Kingdom) and transferred into15×96-well cell culture plates (#353075, Becton Dickinson) with 200μl/well CLONACELL™-HY Medium E. Hybridoma culture media were changed,and 3 days later hybridoma supernatants were screened by enzyme-linkedimmunosorbent assay (ELISA) to assay for binding to muHtrA1-PD-His.

ELISA was performed according to a standard protocol. Briefly, 96-wellmicroliter ELISA plates (Greiner, Germany) were coated with 100 μl/wellmuHtrA1-PD-His or huHtrA1-PD-His (SEQ ID NO: 154) at 2 μg/ml in 0.05 Mcarbonate buffer (pH 9.6) at 4° C. overnight. After washing three timeswith wash buffer (0.05% TWEEN®20 in PBS, Sigma), plates were blockedwith 100 μl ELISA assay diluents with BSA. 100 μl of culturedsupernatants or diluted purified monoclonal antibodies (mAbs) were addedand incubated for 1 h at room temperature. The plates were washed threetimes and incubated with HRP-conjugated goat anti-mouse IgG Fc for 1 h.After washing three times, bound enzyme was detected by addition of 100μl/well of the TMB substrate (BioFX Laboratories) for 5 min. Thereactions were stopped by adding 100 μl/well of stop reagent (BioFXLaboratories), followed by detection of color at absorbance 650 nm(A_(650 nm)). 105 initial ELISA-positive supernatants were identified.After expansion and culturing for 3 days, these clones were rescreenedin a subsequent ELISA which confirmed that 75 out of the 105 clones werestill ELISA-positive for binding to muHtrA1-PD-His (FIGS. 1A and 1B).The majority of these 75 clones also bound to human HtrA1 proteasedomain (FIGS. 1A and 1B).

The 75 ELISA-positive clones were next tested using an in vitro assay toassess the ability of the hybridoma supernatant to inhibit cleavage of asubstrate by the human HtrA1 protease domain (huHtrA1-PD-His; SEQ ID NO:154). The ENZCHEK® Green Fluorescence Protease Assay (ThermoFisherScientific) assay was used. This assay employs a casein derivative thatis heavily labeled with the pH-insensitive green fluorescent BODIPY® FLdye as a substrate. The fluorescence of the BODIPY® FL dye isintramolecularly quenched in the full-length labeled substrate. Cleavageof the substrate by huHtrA1-PD releases fluorescent BODIPY® FL-labeledpeptides, resulting in an increase in fluorescence signal (FIG. 2A).Briefly, the hybridoma supernatant and HtrA1-PD (20 nM hHtrA1-PD; 40 μlhHtrA1-PD to 60 μl supernatant) were incubated in a buffer (50 mM Tris,200 mM NaCl, 0.25% CHAPS, pH 8.0) in a final volume of 200 μl for 20 minat 37° C. 5 μg/ml of the BODIPY® FL-labeled substrate was added, and thefluorescence (milli relative fluorescence units (mRFU)/min) was read for20 min. Using this blocking assay, 10 out of the 75 clones were found toinhibit human HtrA1-PD-mediated substrate cleavage (FIGS. 2B and 2C).FIGS. 3A and 3B show a comparison of the ability of a subset of clonesto inhibit the activity of muHtrA1-PD compared to huHtrA1-PD.

After at least 2 rounds of single cell subcloning by limiting dilution,7 clones with varying characteristics (20E2, 19B12, 12A5, 3A5, 15H6,15E3, and 19G10) were scaled up and the supernatants were collected forantibody purification and further assessment. These clones were chosen,in part, based on the ability to inhibit HtrA1 activity in vitro andbinding to muHtrA3 (19B12). The 7 clones were also tested for theability to detect muHtrA1 in an immunohistochemistry as well as for theability to bind murine HtrA3-PD and murine HtrA4-PD (as assessed byELISA). Table 2 shows a summary of qualitative properties of these 7clones.

TABLE 2 Properties of 7 Final Clones Selected for Antibody PurificationBlocks muHtrA1 Binds Binds hu/muHtrA1-PD IHC Binds Binds Clone IsotypemuHtrA1-PD huHtrA1-PD Substrate Cleavage positive muHtrA3-PD muHtrA4-PD20E2 mIgG2a Yes Yes Yes No No No 19B12 mIgG2a Yes Yes Yes No Slightly No12A5 mIgG2b Yes Yes Yes Yes No Yes 3A5 mIgG2a Yes Yes Yes Yes No No 15H6mIgG2a Yes Yes Yes Yes No No 15E3 mIgG2a Yes No Slightly No Yes Yes19G10 mIgG2a Yes Yes No Yes Yes Yes

The next step was to determine whether the seven antibodies selectedabove inhibited HtrA1-mediated cleavage as measured in a FRET Assay.

The hybridoma supernatants were purified by Protein A affinitychromatography, followed by sterile filtration (0.2 μm pore size, NalgeNunc International, NY, USA), and storage at 4° C. in PBS. The purifiedmonoclonal antibodies were confirmed by ELISA and FACS before furthertesting in functional assays. The isotypes of purified mAbs weredetermined by the ISOSTRIP™ mouse monoclonal antibody isotyping kit(Roche Diagnostics Corporation).

The purified antibodies were tested for the ability to inhibit theactivity of HtrA1 in a FRET-based blocking assay (e.g., H2-Opt assay).In this assay, the ability of the antibody to inhibit the cleavage of afluorescence resonance energy transfer (FRET, also referred to asFörster resonance energy transfer) substrate was determined. The FRETpeptide substrate H2-Opt, which has a molecular weight of 1600 Da,includes the donor Mca (7-methoxycoumarin-4-yl-acetyl) and the acceptor(quencher) Dnp (N-2,4-dinitrophenyl). The full-length sequence of theFRET peptide substrate is (Mca)IRRVSYSF(Dnp)KK (SEQ ID NO: 152). In theintact peptide substrate, the quenching moiety Dnp quenches thefluorescence of the Mca donor. Proteolytic cleavage of the FRET peptidesubstrate separates the fluorophore and quencher, thereby relieving thequenching of Mca fluorescence and resulting in an increased fluorescentsignal (FIG. 4A). The assay was performed using the H2-Opt assayconditions described below in Example 3, Section F. The time-dependentincrease in fluorescence intensity is related to the extent of substratehydrolysis. The antibodies were tested at concentrations of 5 nM, 50 nM,and 500 nM. Five of the purified anti-HtrA1 antibodies (15H6, 19B12,3A5, 12A5, and 20E2) retained the ability to block human and murineHtrA1-PD-mediated substrate cleavage (FIGS. 4B and 4C).

Next, the ability of the purified antibodies to inhibit full-lengthHtrA1-mediated substrate cleavage was tested. The FRET-based blockingassay described in the preceding paragraph was employed using purifiedfull-length (FL) muHtrA1 or huHtrA1 muHtrA1-FL and huHtrA1-FL werepurified as described in Example 2 of WO 2013/055998. The purifiedantibodies, including 15H6 and 19B12, inhibited the activity ofhuHtrA1-FL (FIGS. 5A-5B) and muHtrA1-FL (FIGS. 5C-5D). In this assay,clone 15H6 inhibited huHtrA1-FL with an IC50 of 0.7 nM, which wasapproximately two-fold improved compared to the IC50 of the positivecontrol antibody YW505.94, 15H6 inhibited muHtrA1-FL with an IC50 of 1.1nM, which was almost 5-fold improved compared to the positive controlantibody YW505.94. Clone 19B12 inhibited huHtrA1-FL with an IC50 of 1.4nM, and inhibited muHtrA1-FL with an IC50 of 1.0 nM. The selectedantibodies were sequenced as described below in Section D. The sequencesof these five antibodies are shown in FIG. 6A and FIG. 6B. Twoantibodies, 15H6 and 19B12, were selected for further development basedon their preferred molecular profile including their potency and theirselectivity. The reformatting of these antibodies is described below inSection E.

D. Antibody Sequencing from Hybridoma Clones

i. Cloning Variable Region Gene Sequences from Hybridoma Cells Using5′-Rapid Amplification of cDNA Ends (5′-RACE) in a 96-Well Format

For each hybridoma clone, about 25 μl of log-phase-growing cells(0.5-1.0×10⁵ cells/ml) were transferred from tissue culture plates towells of a 96-well U-bottom plate. 150 μl of cold 1×PBS was then addedto wash the cells before spinning down at 1000 rpm for 5 min. Thesupernatant was removed and the cell pellet was resuspended in 25 μl ofcold 1×PBS.

ii. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Reaction

A master mix consisting of the following was prepared (for 50 wells) inan Eppendorf tube: 2.5 μl of RNASEOUT™ (Invitrogen #10777019), 12.5 μlof 10× Synthesis Buffer (5× SUPERSCRIPT® buffer), 12.5 μl ofdithiothreitol (DTT) (0.1M Invitrogen #P/NY00147), 6.25 μl of dNTPs (10mM Invitrogen #18427-013), 12.5 μl of 2.5% nonylphenoxypolyethoxylethanol (NP-40), 6.25 μl of bovine serum albumin (BSA)at 2 mg/ml (BioLabs #90015), 25 μl of RACE4muHC primer (1:100 in PCRgrade water) (Race 3 kappa primer was substituted for sequencing thelight chain (LC)), 37.5 μl of PCR-grade water, and 10 μl of SUPERSCRIPT®3 enzyme (Invitrogen #18080-093). The Race4muHC degenerate primernucleotide sequence is TTT YTT GTC CAC CKT GGT GCT GC (SEQ ID NO: 139),where Y encodes for C or T, and K encodes for G or T. The Race 3 kappaprimer nucleotide sequence is GTA GAA GTT GTT CAA GAA G (SEQ ID NO:140).

2.5 μl/well of master mix was then transferred to a 96-well PCR reactionplate. 1 μl of cells/well was added to the plate, the plate was spunbriefly for 30 seconds, and then the plate was shaken. The plate was setinto the PCR machine and the program was set to 30 min at 45° C.followed by 30 minutes at 50° C. This plate was labeled as Plate A.

iii. Tailing Reaction

A 10× stock tailing buffer was made with the following ingredients (for50 wells): 5 μl of 1 M MgCl₂, 5 μl of 0.1M DTT, 5 μl of 1 M Tris pH 7.5,10 μl of 100 mM dGTP, and 25 μl of PCR-grade water.

A working solution was made with the following ingredients (for 50wells): 50 μl of the 10× stock tailing buffer, 312.5 μl of PCR-gradewater, and 12.5 μl of 300-unit Terminal Deoxynucleotidyl Transferase(TdT) (Promega #M828A/C).

This working solution was then added to the PCR reaction Plate A at 7.5μl/well. Plate A was then placed into the PCR machine for 1 h at 37° C.followed by 5 min at 65° C. This plate was labeled as Plate A/B todistinguish tailing of this plate.

iv. First PCR Reaction

A master mix consisting of the following was prepared (for 50 wells) ina 15-mL Falcon tube: 1050 μl of PCR-grade water, 500 μl of 5× GC cDNAPCR reaction buffer, 500 μl of GC-melt reagent (Clontech #S1091), 50 μlof the forward primer DC5dn, 50 μl of Race7muHC (Race 2 kappa primer forLC), 50 μl of dNTP (10 mM), and 50 μl of GC ADVANTAGE® polymerase(Clontech #S1088). The Race7muHC degenerate primer nucleotide sequenceis CAR GTC AMD GTC ACT GRC TCA G (SEQ ID NO: 141), where R encodeseither A or G, M encodes either A or C, and D encodes either G or A orT. The Race 2 kappa primer nucleotide sequence is GAG GCA CCT CCA GATGTT AAC (SEQ ID NO: 142).

45 μl of this master mix was then transferred into wells of a new FORreaction plate and 1 μl of the template from Plate A/B was then added.This plate was then placed into the PCR machine and run under aTouchdown PCR method with decreasing annealing temperatures, as listedbelow. This plate was labeled Plate C.

Touchdown PCR method:

96° C. for 4 min

3 cycles of [96° C. for 45 sec, 64° C. for 30 sec, 68° C. for 90 sec], 3cycles of [96° C. for 45 sec, 61° C. for 30 sec, 68° C. for 90 sec], 3cycles of [96° C. for 45 sec, 58° C. for 30 sec, 68° C. for 90 sec], 3cycles of [96° C. for 45 sec, 57° C. for 30 sec, 68° C. for 90 sec], 3cycles of [96° C. for 45 sec, 55° C. for 30 sec, 68° C. for 90 sec]

25 cycles of [96° C. for 45 sec, 52° C. for 30 sec, 68° C. for 90 sec]

68° C. for 5 min, followed by a 4° C. end hold.

Afterwards, 3 μl of PCR product was run on a 2% ethidium bromide (EtBr)E-GEL® and bands were checked. Exol/shrimp alkaline phosphatase (SAP)was added at 10 μl/well and placed in the PCR machine for 45 min at 37°C. followed by 15 min at 85° C., Exol/SAP master mix was made for 50wells: 2.5 μl of 20 unit/μl Exonuclease I (Fermentas #EN0582), 25 μl ofSAP (USB #70092Y), 472.5 μl of PCR-grade water. A 1:4 dilution of thePCR product was made in water. The Race7muHC primer was used forsequencing the heavy chain (HC) and the Race7.1muLC primer was used forsequencing the LC. The Race7.1muLC primer nucleotide sequence is ACT GCTCAC TGG ATG GTG GGA AG (SEQ ID NO: 143).

v. Gel Filtration and Mass Spectrometry Characterization

For gel filtration analysis, 10 ml of purified sample was injected ontoTSK-GEL® Super SW3000 (4.6 mm inner diameter×30 cm, TOSOH Bioscience) at0.35 ml/min using 200 mM K₂PO₄, 250 mM KCl, pH 7.0 as the mobile phase.Approximately 2 mg of purified IgG was reduced with 50 mM dithioreitolat 37° C. for 20 min and analyzed by time-of-flight (TCF) massspectrometry (Agilent LC/MS 6224) after on-line reversed-phaseseparation using a PLRP-S column (Agilent) and acetonitrile gradient.Intact masses were determined by maximum entropy deconvolution ofcollected m/z spectra using MassHunter Qualitative Analysis software(Agilent).

vi. Results

The sequences of the heavy chain variable region (VH) for 19B12, 20E2,3A5, 12A5, and 15H6 are shown in FIG. 6A. The sequences of the lightchain variable region (VL) for 19B12, 20E2, 3A5, 12A5, and 15H6 areshown in FIG. 6B. These clones have unique heavy and light chains. Massspectrometry data corroborated the sequence data (see Tables 3 and 4)(see, e.g., Bos et al. Biotechnol. Bioeng. 112(9): 1832-1842, 2015).

TABLE 3 HC Masses as determined by Mass Spectrometry HC Masses Clone(Daltons) 19B12 51130 50968 20E2 51006 51168 3A5 50684 50846 12A5 5135551193 15H6 50828 50666 50990

TABLE 4 LC Masses as determined by Mass Spectrometry LC Masses Clone(Daltons) 19B12 23970 20E2 23963 3A5 23966 12A5 23895 15H6 23230

E. Reformatting Antibodies 15H6 and 19812

i. TOPO® Cloning of Antibodies 15H6 and 19B12

TOPO® cloning was performed to confirm the variable region sequences ofantibody clones 15H6 and 19B12 obtained from direct sequencing of the5′-RACE PCR products (described above). The TOPO® TA cloning reactionwas done as described in the pCR™4-TOPO® TA Cloning Kit for Sequencing(Invitrogen K4575-02) manual. Briefly, 2 μl of the PCR product, 2 μl ofwater, 1 μl of the pCR™4-TOPO® vector, and 1 μl of the included saltsolution were combined in a tube, mixed, and incubated for 5 min at roomtemperature. The reaction was then placed on ice and 2 μl of this TOPO®Cloning reaction was added to a thawed vial of ONE-SHOT® chemicallycompetent TOP10 Escherichia coli cells (Invitrogen K4575-02) and mixedwithout pipetting. The reaction was incubated on ice for 5-30 min. Cellswere then heat-shocked for 30 sec at 42° C. without shaking. Tubes wereimmediately transferred to ice. 250 μl of room temperature SOC mediumwas added. The tube was capped and shaken horizontally at 200 rpm at 37°C. for 1 h. Next, 50 μl from each transformation was spread onto apre-warmed LB agar plate containing 50 μg/ml of carbenicillin. Plateswere incubated at 37° C. overnight, colonies were picked the next day,and plasmid purification was performed. Sequences were verified andparticular wells containing 15H6 VH and VL and 19B12 VH and VL sequenceseach in a TOPO® vector were selected for use as source vectors inrestriction-free cloning.

ii. Restriction-Free Cloning into mIgG2a

Heavy and light chain variable regions in the TOPO® vectors wereamplified by setting up a PCR mix in the following way for the LC: 0.5μl of template DNA (miniprep source vector), 4 μl of 15H6 VL forwardprimer, 4 μl of 15H6 VL reverse primer, 2 μl of 10 mM dNTPs, 20 μl of 5×HF Buffer, 1 μl of PHUSION® polymerase (F-549L, Thermo Scientific, 2U/μl), and 68.5 μl of water. The reaction mix for cloning the HC was setup the same way except that 15H6 VH forward and reverse primers wereused. 19B12 FOR mixes were set up in the same way as the 15H6 mixesexcept that 19B12 primers were used. The primer sequences were asfollows:

15H6 VL forward primer nucleotide sequence: (SEQ ID NO: 144)GCA ACT GCA ACT GGA GTA CAT TCA CAA ATT GTT CTC TCC CAG TCT CC.15H6 VL reverse primer nucleotide sequence: (SEQ ID NO: 145)GGA TAC AGT TGG TGC AGC ATC AGC CCG TTT GAT TTC CAG CTT GG.15H6 VH forward primer nucleotide sequence: (SEQ ID NO: 146)GCA ACT GCA ACT GGA GCG TAC GCC CAG GTC CAG CTG CAG CAG TCT GG.15H6 VH reverse primer nucleotide sequence: (SEQ ID NO: 147)GGG CCC TTG GTG GAG GCT GAG GAG ACG GTG ACT GAG GTT CCT TGA CCC.19B12 VL forward primer nucleotide sequence: (SEQ ID NO: 148)GCA ACT GCA ACT GGA GTA CAT TCA AAC ATT GTG GTG ACC CAA TCT CC.19B12 VL reverse primer nucleotide sequence: (SEQ ID NO: 149)GGA TAC AGT TGG TGC AGC ATC AGC CCG CTT TAT TTC CAG CTT GG.19B12 VH forward primer nucleotide sequence: (SEQ ID NO: 150)GCA ACT GCA ACT GGA GCG TAC GCC GAG GTG AAG CTG GTG GAA TCT GGG GGA GG.19B12 VH reverse primer nucleotide sequence: (SEQ ID NO: 151)GGG CCC TTG GTG GAG GCT GAG GAG ACG GTG ACT GCG GTT CCT TGA CCC.

The PCR cycling conditions were as follows:

98° C. for 30 seconds

35 cycles of [98° C. for 15 seconds, 68° C. for 30 seconds, 72° C. for35 seconds]

72° C. for 10 minutes

Amplified VH and VL were used as primers to amplify template DNA bysetting up the FOR mix in the following way for 15H6 LC: 1.25 μl oftemplate mIgG2a PRK vector DNA (1:10 dilution of miniprep), 0.5 μl of15H6 VL FOR product (100-200 ng/μl), 1 μl of 10 mM dNTPs, 10 μl of HFBuffer (5×), 1 μl of PHUSION® polymerase, and 35.75 μl of water. The15H6 HC PCR mix was set the same way except that the 15H6 VH PCR productwas used. The 19B12 FOR mixes were set up the same way except that 19B12PCR products were used.

The PCR cycling conditions were as follows:

98° C. for 30 seconds

25 cycles of [98° C. for 15 seconds, 68° C. for 30 seconds, 72° C. for 4minutes]

72° C. for 10 minutes

18 μl of PCR reaction was then transferred to a new tube and digestedwith 2 μl of DpnI (#RO176L, NEB 20,000 U/ml) for 2 h at 37° C., with thetube spun periodically. 30 μl of competent NOVABLUE SINGLES™ competentcells (Novagen, 70181) were transformed with 1 μl of DpnI digestaccording to the manufacturer's instructions. Briefly, cells werethawed, DNA was added, and cells were incubated on ice for 5 min beforebeing heat-shocked for 30 sec, placed back on ice for 2 min, followed byaddition of SOC medium. 25 μl or 50 μl were plated on 50 μg/mlcarbenicillin-containing plates and set at 37° C. overnight. Colonieswere picked the next day, plasmid purification was performed viaminiprep, and plasmids were sequenced.

iii. Antibody Purification

Automated purification from 293 cell supernatants was performed on aTecan Freedom EVO® 200 liquid handling system with a 500 ml MCA96 head.Briefly, IgGs were captured using tip columns that were custom-packedwith 20 ml MABSELECT SURE™ resin (Glygen Corp., Columbia, Md. & GEHealthcare, Pittsburgh, Pa.). After washing with 1×PBS pH 7.4, IgGs wereeluted into 160 ml of 50 mM phosphoric acid, pH 3, and neutralized with12 ml of 20×PBS pH 11. MABSELECT SURE™ tip columns were stripped in 0.1M NaOH and regenerated with 1×PBS pH 7.4 for consecutive use of up to 15times. Similarly, Fabs were captured using tip columns packed with 20 mLGAMMABIND™ Plus resin (Glygen Corp & GE Healthcare) and weresubsequently washed with 1×PBS pH 7.4. Fabs were eluted into 190 ml of10 mM citrate, pH 2.9, and neutralized with 19 ml 0.4 M Tris pH 8.7GAMMABIND™ Plus tip columns were stripped with 6 M guanidine andregenerated with 1×PBS pH 7.4 for consecutive use of up to 15 times.

iv. Recombinant 15H6 and 19B12 Antibodies Retain Original BlockingActivities

The ability of the recombinant 15H6 and 19B12 antibodies to inhibithuHtrA1-FL activity was evaluated using the FRET blocking assaydescribed in Section C above. Both recombinant antibodies retained theiroriginal blocking activities as determined from hybridoma-derivedantibodies (FIGS. 7A and 7B). Recombinant 15H6 antibody had an IC50 ofapproximately 0.7 nM, while recombinant 19B12 antibody had an IC50 ofapproximately 1.2 nM, which mirrored the activity of thehybridoma-derived antibodies (FIGS. 7A and 7B).

Example 2: Humanization of Anti-HtrA1 Hybridoma Antibodies 15H6 and19B12

A. Humanization of Anti-HtrA1 Antibody 15H6

The light chain variable region (VL) and heavy chain variable region(VH) sequences of murine 15H6 antibody (also referred to as m15H6) werealigned with human antibody consensus sequences, and human consensuslight chain kappa I (huκ1) and human consensus heavy chain subgroup I(huVH1) were identified as the closest human frameworks (FIGS. 8A and8B).

The hypervariable regions (HVRs) of the m15H6 light chain and heavychain were grafted into huκI and huVH1 consensus acceptor frameworks,respectively, by Kunkel mutagenesis (see, e.g., Kunkel et al., MethodsEnzymol. 154: 367-382, 1987) using separate oligonucleotides for eachhypervariable region to generate antibody clone h15H6.v1 (FIGS. 8A and8B). In this process, positions 24-34 in HVR-L1, 50-56 in HVR-L2 and89-97 in HVR-L3 of the m15H6 VL were grafted to the huκI consensusacceptor, and positions 26-35 in HVR-H1, 49-65 in HVR-H2, and 95-102 inHVR-H3 of the m15H6 VH were grafted to the huGI consensus acceptor.Positions 46, 47 and 49 in framework region 2 of the light chain(FR-L2), and positions 67, 69, 71, and 93 in framework region 3 of theheavy chain (FR-L3) were also included in the humanization processbecause Foote and Winter have analyzed antibody and antigen complexcrystal structures and found these positions to be part of the frameworkresidues acting as “Vernier” zone, which may adjust HVR structure andfine-tune to fit to antigen (Foote et al., J. Mol. Biol. 224:487-499,1992) (FIGS. 8A-8B). The binding affinity of ml 5H6 and h15H6.v1 in Fabformat for human and murine HrtA1 was measured by BIACORE™ surfaceplasmon resonance (SPR) binding analysis as described in Subsection iiof Section C below (FIGS. 9A-9D). Table 5 summarizes the results of thisanalysis.

TABLE 5 Kinetic Binding Analysis of m15H6 and h15H6.v1 to HtrA1 huHtrA1muHtrA1 Clone k_(on) (1/Ms) K_(off) (1/s) KD (nM) k_(on) (1/Ms) K_(off)(1/s) KD (nM) m15H6 Fab 5.06 × 10⁵ 1.55 × 10⁻⁴ 0.31 5.39 × 10⁵ 1.63 ×10⁻⁴ 0.3 h15H6.v1 Fab 1.17 × 10⁶ 3.59 × 10⁻⁴ 0.31 5.98 × 10⁵  3.2 × 10⁻⁴0.53

To further evaluate the importance of murine Vernier residues inh15H6.v1, this antibody was displayed on phage and individual Verniermurine residues (LC: P46, W47, S49; HC: A67, L69, A71 and T93) werereplaced with human residues (LC: L46, L47, Y49; HC: V67, 169, R71 andA93) to generate point mutation variants. All 7 variants were subject tophage competition ELISA against human or murine HtrA1 to determine thebinding affinities (in terms of phage IC50, see Subsection i of SectionC below) The results indicated that the LC-P46L variant totallyabolished h15H6.v1 binding to both human and murine HtrA1. The LC-S49Yvariant reduced binding with human and murine HtrA1 about 10-fold (FIGS.10A and 10B). The HC variants HC-A67V and HC-T93A both only affectedbinding to murine HtrA1 but not human HtrA1 (FIGS. 10A and 10B). Theother variants, LC-W47L, HC-L69I, and HC-A71R, did not show anysignificant drop in binding to human and murine HtrA1 (FIGS. 10A and10B). Therefore, antibody clone h15H6.v1 was further engineered byadding the following mutations: LC-W47L, HC-L69I, and HC-A71R togenerate antibody clone h15H6.v2 (see FIGS. 8A and 8B).

Chemical stability analysis of antibody clone h15H6.v2 identifiedseveral potentially unstable residues or residue pairs in HVRs: W91 inHVR-L3 (oxidation), N94 P95 in HVR-L3 (clipping), and D55 G56 in HVR-H2(isomerization). See, e.g., Example 4 below. To address W91 oxidation inHVR-L3, 2 variants (LC-W91Y and LC-W91L) were generated and produced asFabs for BIACORE™ SPR binding analysis. The results, summarized in Table6 below, indicated that position LC-W91 is important for bindingaffinity, and the substitutions impacted binding to human and murineHtrA1 by about 20-50 fold (FIGS. 11A and 11B).

TABLE 6 Kinetic Binding Analysis of h15H6.v2 LC-W91 Variants to HtrA1huHtrA1 muHtrA1 Clone k_(on) (1/Ms) K_(off) (1/s) KD (nM) k_(on) (1/Ms)K_(off) (1/s) KD (nM) H15H6.V2 6.9 × 10⁵ 1.2 × 10⁻⁴ 0.2 3.6 × 10⁵ 1.5 ×10⁻⁴ 0.4 H15H6.V2 LC-W91L 1.5 × 10⁵ 1.6 × 10⁻³ 10.7 1.2 × 10⁵ 9.1 × 10⁻⁴7.6 H15H6.V2 LC-W91Y 3.3 × 10⁵ 3.7 × 10⁻³ 11.2 2.4 × 10⁵ 1.7 × 10⁻³ 7.1

For the clipping at positions LC-N94 LC-P95 of HVR-L3, four variants ofh15H6.v2 (LC-N94A LC-P95 (also referred to as AP), LC-N94E LC-P95 (alsoreferred to as EP), LC-N94Q LC-P95 (also referred to as QP), and LC-N94SLC-P95 (also referred to as SP)) were generated and produced as Fabs forBIACORE™ binding analysis. The results indicated that AP and EP variantsshowed similar binding affinity to human HtrA1, and QP and SP variantshad an approximate 2-fold reduction in binding affinity to human HtrA1(FIGS. 12A-12B). A table summarizing the results of this analysis isshown in FIG. 12B.

For the isomerization at residues HC-D55 HC-G56 of HVR-H2, four variants(HC-D55A HC-G56 (also referred to as AG), HC-D55E HC-G56 (also referredto as EG), HC-D55S HC-G56 (also referred to as SG), and HC-D55 HC-G56A(also referred to as DA)) were generated and produced as Fabs forBIACORE™ binding analysis. The results indicated only the EG variantshowed comparable binding affinity against human HtrA1, and the rest ofthe variants at heavy chain positions 55 and/or 56 had a 2- to 3-foldreduction in binding affinity to human HtrA1 (FIGS. 13A and 13B). Atable summarizing the results of this analysis is shown in FIG. 13B.

Based on these results, both of the variants AP (HVR-L3) and EG (HVR-H2)were introduced into the sequence of antibody clone h15H6.v2 to generateantibody clone h15H6.v2.APEG (also referred to as h15H6.v3 or AP_EG)(see FIGS. 8A and 8B). This clone was also compared with several othervariants of h15H6.v2, including LC-N94E LC-P95 HC-D55E HC-G56 (alsoreferred to as EP_EG); LC-N94Q LC-P95 HC-D55E HC-G56 (also referred toas QP_EG); and LC-N94S LC-P95 HC-D55E HC-G56 (also referred to asSP_EG). BIACORE™ SPR analysis indicated that h15H6.v2.APEG retained acomparable binding affinity to h15H6.v2 (FIGS. 14A and 14B). A tablesummarizing the results of this analysis is shown in FIG. 14B. Theability of these variants to block the activity of HtrA1 activity wasdetermined using the FRET-based blocking assay described in Example 1(FIGS. 14C and 14D). The pI of these variants in Fab format was alsodetermined using the software program SMACK (see, e.g., Sharma et al.Proc. Natl. Acad. Sci. USA 111: 18601, 2014) and is shown in Table 7 ascompared to the anti-VEGF Fab ranibizumab (LUCENTIS®).

TABLE 7 pI of H15H6.v2 Variants Antibody Clone pI h15H6.v2.APEG(h15H6.v3) 8.25 EP_EG 7.45 QP_EG 8.25 SP_EG 8.25 Ranibizumab 8.55

B. Humanization of Anti-HrtA1 Hybridoma Antibody 19B12

The amino acid sequences of the VL and VH of murine antibody 19B12 (alsoreferred to as m19B12) were aligned with human consensus sequences, andhuman consensus light chain kappa IV (huκ4) and human consensus heavychain subgroup III (huVH3) were identified as the closest humanframeworks (FIGS. 15A-15B).

The hypervariable regions of the m19B12 light chain and heavy chain weregrafted into huκ4 and huVH3 consensus acceptor frameworks, respectively,by Kunkel mutagenesis using separate oligonucleotides for eachhypervariable region to generate a direct HVR-graft variant, referred toherein as antibody clone h19B12.v1. In this process, positions 24-34 inHVR-L1, 50-56 in HVR-L2 and 89-97 in HVR-L3 of the 19B12 VL were graftedto the huK4 consensus acceptor, and positions 26-35 in HVR-H1, 50-65 inHVR-H2, and 95-102 in HVR-H3 of the 19B12 VH were grafted to the huGIIIconsensus acceptor (FIGS. 15A-15B).

The binding affinity of m19B12 and h19B12.v1 (in Fab format) weredetermined using BIACORE™ SPR (FIGS. 16A-16D) using the approachdescribed in Subsection ii of Section C below. The results of thisanalysis are summarized in Table 8 below. The equilibrium bindingconstant (KD) of h19B12.v1 to human HtrA1 improved approximately 2-foldfollowing humanization (Table 8) as compared to m19B12.

TABLE 8 Kinetic Binding Analysis of m19B12 or h19B12.v1 to HtrA1 huHtrA1muHtrA1 Clone k_(on) (1/Ms) K_(off) (1/s) KD (nM) k_(on) (1/Ms) K_(off)(1/s) KD (nM) m19B12 0.96 × 10⁵ 22.8 × 10⁻⁴ 23.8 1.82 × 10⁵ 11.7 × 10⁻⁴6.43 h19B12.v1  1.5 × 10⁵  1.6 × 10⁻³ 10.7  1.2 × 10⁵  9.1 × 10⁻⁴ 7.6

C. Materials and Methods

i. Phage Competition ELISA to Determine Phage IC50

MAXISORP™ microliter plates were coated with human HtrA1-PD-His at 2μg/ml in PBS for 2 h and then blocked with PBST buffer (0.5% BSA and0.05% TWEEN®20 in PBS) for 1 h at room temperature. Phage purified fromculture supernatants were incubated with serially-diluted human ormurine HtrA1 in PBST buffer in a tissue-culture microtiter plate for 1h, after which 80 μl of the mixture was transferred to humanHtrA1-coated wells for 15 min to capture unbound phage. The plate waswashed with PBT buffer (0.05% TWEEN®20 in PBS), and HRP-conjugatedanti-M13 antibody (Amersham Pharmacia Biotech) was added (1:5000 in PBSTbuffer) for 40 min. The plate was washed with PBT buffer and developedby adding tetramethylbenzidine substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.). The absorbance at 450 nm was plottedas a function of target concentration in solution to determine phageIC50. This was used as an affinity estimate for the Fab clone displayedon the surface of the phage.

ii. Antibody Affinity Determinations by BIACORE™

To determine the binding affinity of anti-HtrA1 Fabs by single-cyclekinetics, (SPR) measurement with a BIACORE™ T100 instrument was used.Briefly, a series S sensor chip CM5 was activated with1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) reagents according to the supplier'sinstructions, and streptavidin (Pierce) was coupled to achieveapproximately 2500 response units (RU), followed by blocking un-reactedgroups with 1 M ethanolamine.

For kinetics measurements, biotinylated human or murine HtrA1-PD-His wasfirst injected at 10 μl/min flow rate to capture approximately 150 RU at3 different flow cells (FC), except for FC1 (which served as areference), and then 5-fold serial dilutions of anti-HtrA1 Fab in HBS-Pbuffer (0.01M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) from low(0.48 nM) to high (300 nM) were injected (flow rate: 30 μl/min) oneafter the other in the same cycle with no regeneration betweeninjections. The sensorgram was recorded and subject to reference andbuffer subtraction before evaluating by BIACORE™ T100 EvaluationSoftware (version 2.0). Association rates (k_(on)) and dissociationrates (k_(off)) were calculated using a simple one-to-one Langmuirbinding model. The equilibrium dissociation constant (KD) was calculatedas the ratio k_(off)/k_(on).

Example 3: Affinity Maturation of Anti-HtrA1 Antibody Clone h15H6.v2

A. h15H6.v2 Affinity Maturation NNK Library Construction and Panning

To further improve the affinity of anti-HtrA1 antibody clone h15H6.v2,phage libraries were constructed from variant h15H6.v2 in Fab-amberformat for monovalent Fab phage display with either light chain HVRresidues (i.e., HVR-L1, HVR-L2, and HVR-L3) or heavy chain HVR residues(i.e., HVR-H1, HVR-H2, and HVR-H3) residues randomized using the NNKdegenerate codon that encodes for all 20 amino acids with 32 codons(see, e.g., Brenner et al., Proc. Natl. Acad. Sci. USA 89(12):5381-5383, 1992). Libraries were designed to allow one NNK mutation ineach of the three light chain or heavy chain HVRs. The resultant libraryDNA was electroporated into E. coli XL1 cells, yielding approximately10⁹ transformants. In some instances, soft randomization libraries andNNK epistasis were employed as described in PCT/US2015/055672. Phagelibraries were incubated with 750 mM NaCl in SUPERBLOCK™ PBS buffer(Pierce) and 0.05% TWEEN® 20 for 30 min and then applied onneutravidin-captured biotinylated HtrA1-His tag for first round panningto reduce non-specific charged interaction between HtrA1 and phage. Inthe subsequent two rounds using decreasing concentration of biotinylatedhuHtrA1-PD-His antigen with 1000× non-biotinylated HtrA1 as competitorin solution to increase the selection stringency. See FIG. 17 for aschematic diagram of the panning strategy.

B. Deep Sequencing of h15H6.v2 Affinity Maturation Libraries

For deep sequencing, phagemid double stranded DNA was isolated from E.coli XL-1 cells carrying phagemid vectors from the initial phage libraryand from the third round of selection. Purified DNA was used as templatefor a limited cycle PCR-based amplification of VL and VH regions usingPHUSION® DNA polymerase (New England Biolabs). PCR products werepurified by agarose gel extraction and clean-up (Qiagen Gel ExtractionKit). Eluted amplicon DNA was used as the basis for deep sequencinglibrary preparation with standard Illumina library preparation methods,using a TRUSEQ™ DNA Sample Prep kit (Illumina). Adapter-ligatedlibraries were subjected to a single cycle of PCR and sequenced on theIllumina MISEQ®, using paired-end sequencing with an insert size of 200bp or 300 bp as appropriate to cover the entire length of the amplicon.

C. Deep Sequencing Analysis of h15H6.v2 Affinity Maturation Libraries

Sequencing data were analyzed using the statistical programming languageR (see, e.g., R Core Team, R: A language and environment for statisticalcomputing, 2013) and the ShortRead package (see Morgan et al.,Bioinformatics 25(19): 2607-2608, 2009). Quality control (QC) wasperformed on identified HVR sequences, where each HVR sequence waschecked for the correct length and was allowed to carry only up to oneNNK mutation and no non-NNK mutation. Position weight matrices weregenerated by calculating the frequency of all mutations of everyrandomized position. Enrichment ratios for each mutation were calculatedby dividing the frequency of a given mutation at a given position in thesorted sample with the frequency of the very same mutation in theunsorted sample, as described previously (Fowler et al., Nature Methods7(9): 741-746, 2010). Heatmaps depicting the enrichment ratios for eachmutation in light chain HVR positions and heavy chain HVR positions areshown in FIGS. 18A and 18B, respectively.

Single mutations from the light chain or heavy chain libraries with highenrichment ratio were selected to synthesize for cloning into amammalian Fab expression construct containing a Flag tag to generateFab-Flag tag fusion proteins. Plasmids encoding the heavy or light chainwere transfected to 293T cells for 30 ml expression and Fabs werepurified with an anti-Flag column.

The purified Fabs containing single mutations were used to determinebinding affinity using BIACORE™ SPR analysis (see Section D below). Theaffinity data for single mutations are summarized in Table 9. The offrates ranged from about 0.0013 to about 0.004, compared with a value ofabout 0.0016 for h15H6.v2. Variant LC3 (LC-N31E) improved the off-rate2- to 3-fold over 15H6.v2.

TABLE 9 Biding Affinity of h15H8.v2 Mutations Identified by DeepScanning Mutagesesis Antibody VL SEQ VH SEQ Variant Mutation k_(on)(1/Ms) k_(off) (1/s) KD (M) ID NO: ID NO: h15H6.v2 7.22E+05 1.58E−042.20E−10 73 77 LC7 LC-S92Y 5.78E+05 1.93E−04 3.35E−10 87 77 HC3 HC-T28K4.56E+05 1.71E−04 3.74E−10 73 95 HC2 HC-T30R 3.66E+05 1.64E−04 4.49E−1073 94 HC1 HC-T30K 3.47E+05 1.65E−04 4.75E−10 73 93 LC3 LC-N31E 2.70E+051.30E−04 4.80E−10 83 77 HC5 HC-M34L 5.11E+05 2.46E−04 4.81E−10 73 97 LC5LC-N53E 4.41E+05 2.12E−04 4.81E−10 85 77 LC4 LC-N53H 4.13E+05 2.13E−045.17E−10 84 77 HC8 HC-Y100I 3.93E+05 2.37E−04 6.03E−10 73 100 HC7HC-A58F 2.71E+05 1.65E−04 6.11E−10 73 99 HC9 HC-A100aP 3.40E+05 2.10E−046.18E−10 73 101 LC6 LC-Q89H 3.90E+05 2.51E−04 6.42E−10 86 77 HC4 HC-T28R3.31E+05 2.12E−04 6.43E−10 73 96 LC2 LC-N31H 2.28E+05 1.90E−04 8.36E−1082 77 LC8 LC-S92K 3.43E+05 3.97E−04 1.16E−09 88 77 LC1 LC-S29R 1.48E+052.29E−04 1.55E−09 81 77 LC9 LC-S93I 1.88E+05 4.04E−04 2.15E−09 89 77 HC6HC-E53A 1.01E+05 2.60E−04 2.58E−09 73 98 LC10 LC-F32V, 2.53E+05 2.13E−038.42E−09 90 77 LC-S92K

D. Combination of Selected Variants for Further Affinity Improvement

Most of the single mutations from the heavy chain or light chain NNKlibrary did not improve the binding affinity to HtrA1 over parentalclone h15H6.v2 (Table 9). The variants with the slowest off rates wereselected from both light chain (LC3, LC4, and LC7) and heavy chain (HC1,HC2, HC3, and HC5) variants to generate combination mutants. Thesecombination mutants included both a variant light chain and a variantheavy chain. BIACORE™ kinetic analysis was performed (as described belowin Section C) using the combination mutants. Combination mutantsLC3.HC3, LC3.HC1, and LC7.HC2 had 2-3 fold improved affinity improvementover the parental Fab (h15H6.v2) (Table 10).

TABLE 10 Kinetic Analysis of Combination Mutant Binding to HtrA1 VL SEQVH SEQ Sample k_(on) (1/Ms) k_(off) (1/s) KD (M) ID NO: ID NO: LC3.HC38.01E+05 4.93E−05 6.16E−11 83 95 LC3.HC1 8.85E+05 7.95E−05 8.89E−11 8393 LC7.HC2 1.97E+06 1.89E−04 9.55E−11 87 94 LC3.HC5 5.77E+05 1.02E−041.76E−10 83 97 h15H6.v2 7.22E+05 1.58E−04 2.20E−10 73 77 LC3.HC23.62E+05 1.09E−04 3.02E−10 83 94 LC7.HC3 7.09E+05 2.37E−04 3.35E−10 8795 LC5.HC2 2.94E+05 2.11E−04 7.19E−10 85 94 LC7.HC5 4.06E+05 3.03E−047.45E−10 87 97 LC7.HC1 3.47E+05 2.67E−04 7.72E−10 87 93 LC4.HC1 2.68E+052.28E−04 8.52E−10 84 93 LC5.HC5 3.37E+05 2.96E−04 8.77E−10 85 97 LC31.27E+05 1.15E−04 9.05E−10 83 77 LC5.HC3 2.33E+05 2.51E−04 1.08E−09 8595 LC5.HC1 2.08E+05 2.30E−04 1.10E−09 85 93 LC7 1.37E+05 1.88E−041.37E−09 87 77 LC4.HC5 2.12E+05 2.97E−04 1.40E−09 84 97 LC4.HC3 1.10E+052.40E−04 2.18E−09 84 95 LC4.HC2 2.78E+04 2.22E−04 7.98E−09 84 94

Next, LC mutants (LC37=LC3_LC7, LC347=LC3_LC4_LC7) and HC mutants(HC13=HC1_HC3, HC23=HC2_HC3) were further combined. These mutants werefurther modified by incorporating the HVR-L3 N94A and HVR-H2 D55Evariants (i.e., AP_EG, see Example 2) to avoid self-cleavage anddeamidation for affinity kinetic analysis.

APEGIC3.HC1, APEG.LC3.HC3 (h15H6.v4), APEGIC37.HC13, APEGIC37.HC23,APEGIC347.HC13, and APEG.LC347.HC23 were the top clones, with 3- to5-fold improvements, on average, over h15H6.v2 (FIG. 20). The VL and VHamino acid sequences of these variants are shown in FIGS. 21A and 21B,respectively. The sequences of these clones were analyzed by potentialrisk of oxidation on HVR-L3 W91 using in silico analysis (Sharma et al.Proc. Natl. Acad. Sci. USA 111:18601, 2014). APEG.LC3.HC3 (h15H6.v4),APEGIC37.HC13, and APEGIC347.HC13 were ranked as equivalent risk ash15H6.v3. Others have higher risk than h15H6.v2.

E. Fab Affinity Determination by BIACORE™ SPR

To determine the binding affinity of selected Fab variants for HtrA1,SPR measurement with a BIACORE™ T200 instrument was performed. Briefly,a series S sensor chip CM5 was activated with 1-EDC and NHS reagentsaccording to the supplier's instructions, and anti-His antibody wascoupled to achieve 200-300 response units (RU), then following byblocking un-reacted groups with 1 M ethanolamine. For kineticsmeasurements, approximately 5 nM of huHtrA1-PD-His was first injected at10 μl/min flow rate to capture approximately 100 RU at 2 different flowcells (FC), except for FC1 (which served as a reference). Next, 5-foldserial dilutions of Fab in HBS-P buffer (0.01M HEPES pH 7.4, 0.15M NaCl,0.005% surfactant P20) from low (0.8 nM) to high (50 nM) were injected(flow rate: 30 μl/min). The binding responses on HtrA1 were corrected bysubtracting of RU from a blank flow cell. The sensorgram was recordedand subject to reference and buffer subtraction before evaluating byBIACORE® T200 Evaluation Software (version 2.0). Association rates(k_(on)) and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model. The equilibrium dissociation constant(KD) was calculated as the ratio of k_(off)/k_(on).

F. inhibitory Activity of h15H6.v2 Variants in an HtrA1 FRET-BasedBlocking Assay (H2-Opt Blocking Assay)

Selected affinity matured h15H6.v2 variants were tested for the abilityto inhibit HtrA1 activity. In vitro FRET-based blocking assays using theH2-Opt substrate ((Mca)IRRVSYSF(Dnp)KK) were performed. The H2-Optblocking assays were performed as described in Example 3 of U.S. PatentApplication Publication No. 2013/0129743A1, which is incorporated byreference herein in its entirety. Briefly, the peptide H2-Opt(Mca-IRRVSYSF(Dnp)KK) (SEQ ID NO: 152), originally described as asubstrate for HtrA2 (see, e.g., Martins et al., J. Biol. Chem,278:49417-27, 2003), was synthesized on Fmoc-Lys(Boc)-wing resin usingstandard coupling procedures with HBTU. Fmoc-Lys(DNP)-H (Anaspec) wasincorporated in the P5′ position. The peptide was synthesized up to P5(Mca, 7-Methoxycoumarin, Aldrich) and then cleaved from the solidsupport using trifluoroacetic acid, triisoproplysilane and water for 2hours at room temperature. Peptide was precipitated from ethyl ether,extracted with acetic acid, acetonitrile, water and lyophilized. Crudelabeled peptide was dissolved and purified on preparative reverse phaseC18 column using acetonitrile/water. Purified fractions were pooled,lyophilized, and analyzed by liquid chromatography/mass spectrometry(PE/Sciex) and found to be consistent with their calculated masses.

HtrA1 was incubated in 96-well black optical bottom plates (Nalge NuncInt., Rochester, N.Y.) with anti-HtrA1 antibodies serially diluted inassay buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.25% CHAPS) for 20min at 37° C. A 10 mM stock solution of the peptide substrateMca-IRRVSYSF(Dnp)KK (SEQ ID NO: 152) (H2-Opt) in DMSO was diluted inwater to 12.5 μM, pre-warmed at 37° C. and then added to the reactionmixture. The increase of fluorescence signal (excitation 328 nm,emission 393 nm) was measured on a SPECTRAMAX® M5 microplate reader(Molecular Devices, Sunnyvale, Calif.) and the linear rates of H2-Optcleavage (mRFU/min) determined.

FIG. 22A shows a summary of the results of three independent H2-Optassay experiments performed essentially as described above. Most of theclones had IC50 values in the range of about 0.4 nm to about 0.5 nM, ascompared to about 0.39 nM for h15H6.v2. The anti-HtrA1 antibodyAPEG.LC3.HC3 (also referred to as h15H6.v4) showed the best IC50 valueat about 0.386 nM in this assay (FIG. 22A). FIG. 22B shows arepresentative plot from this analysis. In general, the affinity maturedh15H6.v2 variants showed improved maximal inhibition of HtrA1 comparedto h15H6.v2 (FIG. 22A), with the maximal inhibition values ranging fromabout 78% to about 96%.

The FRET-based H2-Opt blocking assay was used to evaluate the ability ofdifferent antibody formats of the h15H6.v2 variants, including h15H6.v4,to inhibit HtrA1 activity. The H2-Opt assay was performed essentially asdescribed above, except that the assay conditions were: 1 nM huHtrA1-PDenzyme, 1.25 μM H2-Opt substrate, 50 mM Tris pH 8, 200 mM NaCl, 0.25%CHAPS. The results were analyzed based on the relative fluorescent units(RFU)/s rate (50-1000 s) or the endpoint RFU values at 2000 s. FIGS.23A-23H show the results of an exemplary independent experimentcomparing the ability of h15H6.v2 in Fab format, and h15H6.v4 in IgG orFab formats, to inhibit the activity of huHtrA1-PD. The anti-HtrA1antibody YW505.94a (see WO2013/055988) served as a positive control.FIGS. 24A and 24B show a summary of the IC50 and IC90 results,respectively, from a first set of three independent experiments analyzedusing the RFU/s approach. FIGS. 25A and 25B show a summary of the IC50and IC90 results, respectively, from a second set of three independentexperiments analyzed using the RFU/s or the endpoint RFU approach forh15H6.v4 Fab.

G. Blocking Ability of APEG.LC3.HC3 (h15H6.v4) in a MassSpeclrometry-Based Activity Assay

The ability of APEG.LC3.HC3 (h15H6.v4) to inhibit huHtrA1-PD-mediatedcleavage of an intact full-length substrate (α-casein) was assessedusing a mass spectrometry (MS)-based activity assay. In this example,the anti-HtrA1 antibody h15H6.v4 was compared to a small moleculeinhibitor of HtrA proteases, ucf-101, which has been described as anantagonist to HtrA2 (also known as Omi) (Cilenti et al., J. Biol. Chem.278(13):11489-11494, 2003). Tandem Mass Tag (TMT) isobaric mass tagginglabels were employed for MS-based quantitation of α-casein cleavage byHtrA1 in the presence of h15H6.v4 or ucf-101. α-casein has three P1′residues that can be cleaved by HtrA1: Ser72, Thr95, and Ser157.

TMTDUPLEX™ isobaric mass tagging reagents were used to differentiallylabel α-casein standards and assay samples for quantitation of intactα-casein. The TMTDUPLEX™ reagents are sets of isobaric compounds (i.e.,same mass and structure, also called isotopomers) that are NHS-activatedfor covalent, irreversible labeling of primary amines (—NH₂) groups.Each isobaric reagent contains a different number of heavy isotopes inthe mass reporter tag moiety, which results in a unique reporter massduring tandem MS/MS for sample identification and relative quantitation.

100 mM triethylammonium bicarbonate, 100 nM huHtrA1, 100 μg/mL α-casein,and inhibitor (i.e., ucf-101 or h15H6.v4) were incubated (final volume20 μL) at 37° C. for 18 hours (digested solution). Separately, a similarsolution was generated without inhibitor and incubated identically(control solution). h15H6.v4 Fab was tested at concentrations rangingfrom 3.12 nM to 100 nM, while the positive control small moleculeinhibitor ucf-101 was tested at concentrations ranging from 2 nm to 250nM.

Tandem mass tag (TMTDUPLEX™) stock solutions were generated asrecommended by vendor (Thermo Fisher Scientific). 5 μL of TMT-126 wasadded to the digested solution and 5 μL of TMT-127 was added to thecontrol solution. After 1 h incubation at room temperature, the abovesolutions were combined on an equal volume basis. The samples were runon LC-MS and quantitated by fragmentation of most intense ion peaksusing higher-energy C-trap dissociation (HC©); the reporter ionintensity ratio of 126/127 after fragmentation was used to determine theconcentration in the digested solution.

A titration curve showed that the assay accurately quantified intactα-casein (FIG. 26B). In this assay, h15H6.v4 Fab inhibited thehuHtrA1-PD-mediated cleavage of intact α-casein with an IC50 of about 45nM (IC90=about 71 nM). This value was markedly improved compared to thesmall molecule inhibitor ucf-101, which had an IC50 of 77 μM.

H. Blocking Ability of h15H6.v2 Affinity Matured Variants in anEndogenous HtrA1 Activity Assay

The ability of h15H6.v2 affinity matured variants to inhibit endogenousHtrA1 activity in a rabbit eye model was assessed. For the endogenousHtrA1 activity assay, media from HtrA1-secreting cells (human C32melanoma cells) was used as the source of HtrA1. See, e.g., Ciferri etal. Biochem J. Sep. 18, 2015, DOI: 10.1042/BJ20150601. Note that in theendogenous assay, there is an approximate 10-fold higher concentrationof HtrA1 compared to the recombinant HtrA1 H2-Opt assay (see, e.g., FIG.28), which is considered to explain the different IC50 values observedin the H2-Opt assays using endogenous HtrA1 and recombinant HtrA1. FIGS.27A-27C show results from the Endogenous HtrA1 Assay. In particular,clone APEG.LC3.HC3 (h15H6.v4) had an IC50 of about 1.125 nM (FIG. 27C),with a maximal inhibition of about 80.1%.

I. Summary of Properties for Selected h15H6.v2 Variants

The kinetic binding and inhibitory activity of selected derivatives ofthe anti-HtrA1 antibody clone h15H6.v2 were compared using BIACORE SPRanalysis and the FRET-based H2-Opt activity assay. To determine maximalinhibition, positive and negative controls were used to determine 0%inhibition (enzyme only, no inhibitor) and 100% inhibition (no enzyme).The results of this comparison are shown in FIG. 28.

Example 4: Molecular Assessment Analysis of Anti-HtrA1 Antibodies

Anti-HtrA1 antibody clones h15H6.v2, h15H6.v2.APEG (also referred to ash15H6.v3), and APEG.LC3.HC3 (also referred to as h15H6.v4) were testedin molecular assessment (MA) analyses for stability properties. Theanti-HtrA1 antibody clone h19B12.v1 was also tested. Briefly, theanti-HtrA1 antibodies (1 mg/ml) were tested for stress under chemicalconditions with AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), asmall molecule known to generate free radicals (see, e.g., Ji et al., J.Pharm. Sci. 98(12):4485-4500, 2009), as well as under thermal conditionsat varying pH (a two-week thermal stress test at 40° C., pH 5.5) (see,e.g., Zhang et al., J. Chromatography A 1272:56-64, 2013).

Table 11 shows a comparison between the results of MA analyses forh15H6.v2 and h15H6.v3. Notably, LC-W91 in HVR-L3 had increased oxidationfollowing AAPH stress in both h15H6.v2 and h15H6.v3 (about 84.5% andabout 86.4%, respectively). Table 12 shows results of MA analysis forh15H6.v4. Surprisingly, the oxidation at LC-W91 in HVR-L3 for h15H6.v4was reduced compared to h15H6.v2 and h15H6.v3, with only a 26.5%increase in oxidation following AAPH stress compared to approximately84.5-86.4% increase in oxidation for h15H6.v2 and h15H6.v3. Thisimprovement was unexpected because APEG.LC3.HC3 has only twosubstitutions compared to h15H6.v3, i.e., HC-T28K in the FR-H1 regionand LC-N31E in HVR-L1, both of which were introduced to improve affinityand neither of which was expected to impact oxidation of LC-W91. Theh15H6.v4 antibody used in this MA analysis was prepared using asingle-column purification procedure. When the MA analysis for h15H6.v4was repeated using antibody prepared using a two-column purificationprocedure, LC-W91 was shown to be unstable under AAPH stress. It isbelieved that the results of the AAPH stress assessment performed withmaterial purified using the two-column purification was different fromthe results obtained using material purified by a single-columnpurification procedure because the single-column purified materialcontained a contaminant that interfered with the AAPH stress assessment.

TABLE 11 MA Properties of h15H6.v2 and h15H6.v3 Stress h15H6.v2 h15H6.v3Thermal D³¹S³² in HVR-H1 is stable Not determined Stress D⁵⁵G⁵⁶ inHVR-H2 is unstable - 5.8% increase in isomerization D⁵²P^(52a) in HVR-H2is stable AAPH M³⁴ in HVR-H1 is stable M in HVR-H1 is stable Stress W⁹¹in HVR-L3 is unstable - 84.5% W in HVR-H3 is stable increase inoxidation W⁹¹ in HVR-L3 is (3.7% in control and 88.2% in unstable -86.4% AAPH) W⁹⁶ in HVR-L3 is stable increase in oxidation (0.1% incontrol and 86.5% in AAPH) W⁹⁶ in HVR-L3 is stable Size Monomer loss(0.9%) is acceptable Not determined Charge Main peak loss (14.3%) isacceptable Not determined LC/MS Masses are as expected Not determined

TABLE 12 MA Properties of h15H6.v4 (APEG.LC3.HC3) Stress h15H6.v4(APEG.LC3.HC3) Thermal D³¹S³² in HVR-H1 is stable Stress D⁵²P^(52a) inHVR-H2 is stable D⁹⁷Y⁹⁸ in HVR-H3 is stable D⁹⁹Y¹⁰⁰ in HVR-H3 is stableD¹⁰¹Y¹⁰² in HVR-H3 is stable AAPH M³⁴ in HVR-H1 is stable Stress W⁹¹ inHVR-L3 is stable - 26.5% increase in oxidation (0.5% in control and27.0% in AAPH stress) W⁹⁶ in HVR-L3 is stable Size Monomer loss (0.1%)is acceptable Charge Main peak loss (3.7%) is acceptable LC/MS Massesare as expected

Table 13 shows the results of MA analysis for the anti-HtrA1 antibodyclone h19B12.v1. HC-N52a HC-G53 in HVR-H2 were determined to beunstable, with a 49% increase in deamidation. Accordingly, substitutionsat these HVR-H2 positions of h19B12.v1 (e.g., HC-N52aE, HC-N52aS,HC-N52aS, and HC-N52a HC-G53A) are expected to improve stability.

TABLE 13 MA Properties of h19B12.v1 Stress h19B12.v1 Thermal D⁶¹T⁶² inHVR-H2 is stable Stress D¹⁰⁰G^(100a) in HVR-H3 is stable D^(27c)S^(27d)in HVR-L1 is stable N⁹¹N⁹² in HVR-L3 is stable D⁹⁴P⁹⁵ in HVR-L3 isstable AAPH N^(52a)G⁵³ in HVR-H2 is unstable (49% Stress change indeamidation) (t₀: 15% to t_(4wk): 64%) M^(100d) in HVR-H3 is stable M³³in CDR-L1 is stable M³⁴ in CDR-H1 is stable Size Monomer loss (1.8%) isacceptable Charge Main peak loss (56.3%) is unacceptable LC/MS Massesare as expected

Example 5: Structure of h15H6.v4 Fab Bound to HtrA1

The structure of h15H6.v4 Fab bound to HtrA1 was determined by X-raycrystallography and electron microscopy. The results demonstrated thatthe h15H6.v4 Fab HtrA1 epitope is formed primarily by amino acids thatcomprise the turn of the LA loop of HtrA1.

Peptide Synthesis:

Peptides corresponding to regions of the HtrA1 protein were generatedusing methods well known in the art. See, for example, Atherton, E., etal. (1978). J. Chem Soc. Chem. Commun. 13:537-539.

Crystallization:

The h15H6.v4 Fab (1 mL) at 10 mg/ml in 0.15M NaCl, 20 mM Tris pH 7.5 wasincubated overnight at 4° C. with 1 mg of peptide (3 fold molar excesspeptide/protein) containing amino acids in the LA loop of HtrA1. TheFab-peptide complex was crystallized using 2M ammonium sulfate, 0.2Mpotassium acetate.

X-Ray Refinement:

An h15H6.v4 Fab/HtrA1 peptide crystal was harvested and preserved fordiffraction analysis by immersion in a cryo-protectant solution madefrom addition of 30% glycerol to mother liquor followed by suddenimmersion in liquid nitrogen. Data were collected at SSRL beamline 12-2and processed using XDS (Kabasch W (2010) Acta Crystallogr D BiolCrystallogr. 266:125-32). Molecular replacement for the Fab-peptidecomplex was achieved using the Fab structure as a search probe in CCP4(Winn M D et al. (2011) Acta Crystallogr D Biol Crystallogr. 67:235-42).After rigid body refinement, Fo-Fc density could be seen for thepeptide. A portion of the HtrA1 protease domain residues from loop A(RKLPFSKREVPV) (PDB 3TJO) were then fit into the density essentially asdescribed in Emsley et al. (2010) Acta Crystallogr D Biol Crystallogr.66:125-32.

Several rounds of refinement in Phenix (Adams P D et al. (2010) ActaCrystallogr D Biol Crystallogr. 66:213-21) were utilized. A final roundof refinement in Buster (Bricogne G et al. (2011) brought the R valuesto R=16.8%, Rfree=19.8% 2.1 Å resolution.

Electron Microscopy (EM) structure of Fab15H6.v4 bound to HtrA1

For EM imaging, 4 μl of HtrA1-hFab15H6.v4 complex was incubated for 30sec on a freshly glow discharged continuous carbon 400-mesh copper grid(Electron Microscopy Sciences). After incubation, the sample wasnegatively stained using a solution of 2% (w/v) uranyl formate (SPISupplies). Excess stain solution was blotted away with Watman paper andthe grid was air-dried. HtrA1-Fab15H6.v4 sample was analyzed on aTecnai-12 BioTween (FEI) equipped with a LaB6 filament and operated at120 keV under low dose conditions. Images were collected using a 4 k×4 kCCD camera (Gatan Inc.) at a nominal magnification of ×62,000 (2.22 Åper pixel). 27346 particles, having a box size of 128 px weresemi-automatically picked, using the swarm algorithm available under thee2dogpicker.py routine included into EMAN2 distribution (Tang L et al.(2007) J. Chem. Inf. Model. 47:1438-45). These particles were thensubjected to reference free 2D classification using the software suiteRelion (Scheres S H (2012) J. Struct. Biol. 180:519-30). Given theflexibility between the HTRA1 trimer and the bound Fabs, the 3Dclassification algorithm of Relion was used to generate five 3D volumesusing as a starting model the crystal structure of HtrA1 trimer (PDB ID3TJO) low pass filtered to a resolution of 60 Å. Each of these volumeswas finally refined using the Refine3D algorithm in Relion. Atomicdensities of the HTRA1 trimer and the crystal structure of theFab15H6.v4 were fitted into the EM density using the fit in mapalgorithm in Chimera (Pettersen E F et al., 2004). J. Comput. Chem.25:1605-12 (2004).

Validation of Structure of Fab15H6.v4 Fab Bound to HtrA1 Using AlanineSubstitutions

The structural studies described above demonstrated that the Fab15H6.v4Fab interacts closely with loop “A” of the HtrA1 protein. To confirmthis, a peptide corresponding to residues 190-201 of the human HtrA1sequence (where the numbering corresponds to the numbering of theprecursor protein) was synthesized and tested for binding to h15H6.v4Fab using surface plasmon resonance (SPR) as described above. Thepeptide (LA-pep1) showed a strong binding interaction with 15H6.v4 Fabhaving a KD value of 0.4 nM. See Table 14.

Alanine substitutions in this peptide sequence reduce (LA-pep2, LA-pep5)or completely abolish (LA-pep3, LA-pep4, LA-pep5) the binding to 15H6.v4Fab. These biophysical results are consistent with the structuralstudies described above and in FIGS. 32A and 32B and demonstrate thatthe binding epitope for 15H6.v4 is formed primarily by amino acids thatcomprise the turn of LA Loop of HtrA1.

In contrast, the YW505.94 Fab did not bind to LA-pep1, nor to any of themutant forms (Table 14). This indicates that, despite the fact that theYW505.94 Fab and the h15H6.v4 compete with each other to bind to HtrA1,the epitopes of these two Fabs are distinct. The YW505.94 Fab epitope iscentered at loops B and C, whereas the epitope of 15H6.v4 Fab mainlycomprises the tip of the LA loop.

Although it is not intended that the present invention be bound by anyparticular mechanism, it is possible that the close interaction betweenh15H6.v4 and the LA loop of HtrA1 accounts for the significantlyimproved affinity and potency of this antibody when compared withYW505.94.

TABLE 14 HtrA1 Loop A (LA) peptides binding to 15H6.v4 Faband to YW505.94 Fab h15H6.vr h15H6.v4 Fab YW505.94 AA Fab Affinity FabName Peptide change K_(D) (nM) loss** K_(D) (nM) LA- RKLPFSKREVPV pep1LA-

KLPFSKREVPV R1A 321 810 NB pep2 LA- RK

PFSKREVPV L3A NB* >2,500 NB pep3 LA- RKL

FSKREVPV P4A NB >2,500 NB pep4 LA- RKLP

SKREVPV F5A 4.53 11 NB pep5 LA- RKLPFSK

EVPV R8A NB >2,500 NB pep6 *NB - no binding detected up to 1 μM.**affinity loss = K_(D) mutant/K_(D) wild-type

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. A method of treating an HtrA1-associated disorderor an ocular disorder in a human subject in need thereof, the methodcomprising administering a therapeutically effective amount of anantibody that specifically binds HtrA1, wherein the antibody comprises abinding domain comprising the following six HVRs: (a) HVR-H1 comprisesthe amino acid sequence of DSEMH (SEQ ID NO: 7); (b) HVR-H2 comprisesthe amino acid sequence of GVDPETEGAAYNQKFKG (SEQ ID NO: 8); (c) HVR-H3comprises the amino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d)HVR-L1 comprises the amino acid sequence of RASSSVEFIH (SEQ ID NO: 9);(e) HVR-L2 comprises the amino acid sequence of ATSNLAS (SEQ ID NO: 10);and (f) HVR-L3 comprises the amino acid sequence of QQWSSAPWT (SEQ IDNO: 11).
 2. The method of claim 1, wherein the HtrA1-associated disorderor the ocular disorder is age-related macular degeneration (AMD),diabetic retinopathy, retinopathy of prematurity, or polypoidalchoroidal vasculopathy.
 3. The method of claim 2, wherein theHtrA1-associated disorder is early dry AMD, intermediate dry AMD, oradvanced dry AMD.
 4. The method of claim 3, wherein the advanced dry AMDis geographic atrophy.
 5. The method of claim 1, further comprising thestep of administering a Factor D antagonist, wherein the Factor Dantagonist is an anti-Factor D antibody.
 6. The method of claim 5,wherein the anti-Factor D antibody is lampalizumab.
 7. The method ofclaim 1, wherein the antibody is administered intravitreally, ocularly,intraocularly, juxtasclerally, subtenonly, superchoroidally, ortopically.
 8. The method of claim 7, wherein the antibody isadministered intravitreally by injection.
 9. The method of claim 1,wherein the antibody is administered by a long-acting delivery system.10. The method of claim 9, wherein the long-acting delivery system is aPLGA-based solid implant or an implantable port delivery system.
 11. Amethod of treating an HtrA1-associated disorder or an ocular disorder ina human subject in need thereof, the method comprising administering tothe human subject a therapeutically effective amount of an antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising the following six HVRs: (a) an HVR-H1 comprising theamino acid sequence of DSEX₁H (SEQ ID NO: 1), wherein X₁ is Met or Leu;(b) an HVR-H2 comprising the amino acid sequence of GVDPETX₂GAAYNQKFKG(SEQ ID NO: 2), wherein X₂ is Glu or Asp; (c) an HVR-H3 comprising theamino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASSSVX₃FIH (SEQ ID NO: 4),wherein X₃ is Glu or Asn; (e) an HVR-L2 comprising the amino acidsequence of ATSX₄LAS (SEQ ID NO: 5), wherein X₄ is Asn, His or Glu; and(f) an HVR-L3 comprising the amino acid sequence of QQWX₅SX₆PWT (SEQ IDNO: 6), wherein X₅ is Ser or Tyr and X₆ is Ala or Asn.
 12. A method oftreating an HtrA1-associated disorder or an ocular disorder in a humansubject in need thereof, the method comprising administering to thehuman subject a therapeutically effective amount of an antibody thatspecifically binds HtrA1, wherein the antibody comprises a heavy chainvariable (VH) domain comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 21 and alight chain variable (VL) domain comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO:
 22. 13. A method of treating an HtrA1-associated disorder or anocular disorder in a human subject in need thereof, the methodcomprising administering to the human subject a therapeuticallyeffective amount of an antibody that specifically binds HtrA1, whereinthe antibody comprises a VH domain comprising an amino acid sequencehaving at least 96% sequence identity to the amino acid sequence of SEQID NO: 21 and a VL domain comprising an amino acid sequence having atleast 96% sequence identity to the amino acid sequence of SEQ ID NO: 22.14. A method of treating an HtrA1-associated disorder or an oculardisorder in a human subject in need thereof, the method comprisingadministering to the human subject a therapeutically effective amount ofan antibody that specifically binds HtrA1, wherein the antibodycomprises a VH domain comprising an amino acid sequence having at least97% sequence identity to the amino acid sequence of SEQ ID NO: 21 and aVL domain comprising an amino acid sequence having at least 97% sequenceidentity to the amino acid sequence of SEQ ID NO:
 22. 15. A method oftreating an HtrA1-associated disorder or an ocular disorder in a humansubject in need thereof, the method comprising administering to thehuman subject a therapeutically effective amount of an antibody thatspecifically binds HtrA1, wherein the antibody comprises a VH domaincomprising an amino acid sequence having at least 98% sequence identityto the amino acid sequence of SEQ ID NO: 21 and a VL domain comprisingan amino acid sequence having at least 98% sequence identity to theamino acid sequence of SEQ ID NO:
 22. 16. A method of treating anHtrA1-associated disorder or an ocular disorder in a human subject inneed thereof, the method comprising administering to the human subject atherapeutically effective amount of an antibody that specifically bindsHtrA1, wherein the antibody comprises a VH domain comprising an aminoacid sequence having at least 99% sequence identity to the amino acidsequence of SEQ ID NO: 21 and a VL domain comprising an amino acidsequence having at least 99% sequence identity to the amino acidsequence of SEQ ID NO:
 22. 17. A method of treating an HtrA1-associateddisorder or an ocular disorder in a human subject in need thereof, themethod comprising administering to the human subject a therapeuticallyeffective amount of an antibody that specifically binds HtrA1, whereinthe antibody comprises a VH domain comprising the amino acid sequence ofSEQ ID NO: 21 and a VL domain comprising the amino acid sequence of SEQID NO:
 22. 18. A method of treating an HtrA1-associated disorder or anocular disorder in a human subject in need thereof, the methodcomprising administering to the human subject a therapeuticallyeffective amount of an antibody that specifically binds HtrA1, whereinthe antibody comprises a heavy chain amino acid sequence of SEQ ID NO:160 and a light chain amino acid sequence of SEQ ID NO:
 159. 19. Amethod of treating geographic atrophy in a human subject in needthereof, the method comprising administering to the human subject atherapeutically effective amount of an antibody that specifically bindsHtrA1, wherein the antibody comprises a binding domain comprising thefollowing six HVRs: (i) an HVR-H1 comprising the amino acid sequence ofDSEX₁H (SEQ ID NO: 1), wherein X₁ is Met or Leu; (ii) an HVR-H2comprising the amino acid sequence of GVDPETX₂GAAYNQKFKG (SEQ ID NO: 2),wherein X₂ is Glu or Asp; (iii) an HVR-H3 comprising the amino acidsequence of GYDYDYALDY (SEQ ID NO: 3); (iv) an HVR-L1 comprising theamino acid sequence of RASSSVX₃FIH (SEQ ID NO: 4), wherein X₃ is Glu orAsn; (v) an HVR-L2 comprising the amino acid sequence of ATSX₄LAS (SEQID NO: 5), wherein X₄ is Asn, His or Glu; and (vi) an HVR-L3 comprisingthe amino acid sequence of QQWX₅SX₆PWT (SEQ ID NO: 6), wherein X₅ is Seror Tyr and X₆ is Ala or Asn.
 20. The method of claim 19, furthercomprising the step of administering a Factor D antagonist, wherein theFactor D antagonist is an anti-Factor D antibody.
 21. The method ofclaim 20, wherein the anti-Factor D antibody is lampalizumab.
 22. Amethod of treating wet AMD in a human subject in need thereof, themethod comprising administering to the human subject a therapeuticallyeffective amount of an antibody that specifically binds HtrA1, whereinthe antibody comprises a binding domain comprising the following sixHVRs: (i) an HVR-H1comprising the amino acid sequence of DSEX₁H (SEQ IDNO: 1), wherein X₁ is Met or Leu; (ii) an HVR-H2 comprising the aminoacid sequence of GVDPETX₂GAAYNQKFKG (SEQ ID NO: 2), wherein X₂ is Glu orAsp; (iii) an HVR-H3 comprising the amino acid sequence of GYDYDYALDY(SEQ ID NO: 3); (iv) an HVR-L1 comprising the amino acid sequence ofRASSSVX₃FIH (SEQ ID NO: 4), wherein X₃ is Glu or Asn; (v) an HVR-L2comprising the amino acid sequence of ATSX₄LAS (SEQ ID NO: 5), whereinX₄ is Asn, His or Glu; and (vi) an HVR-L3 comprising the amino acidsequence of QQWX₅SX₆PWT (SEQ ID NO: 6), wherein X₅ is Ser or Tyr and X₆is Ala or Asn.
 23. The method of claim 22, further comprising the stepof administering a Factor D antagonist, wherein the Factor D antagonistis an anti-Factor D antibody.
 24. The method of claim 23, wherein theanti-Factor D antibody is lampalizumab.
 25. A method of treatinggeographic atrophy in a human subject in need thereof, the methodcomprising administering to the human subject a therapeuticallyeffective amount of an antibody that specifically binds HtrA1, whereinthe antibody comprises a VH domain comprising the amino acid sequence ofSEQ ID NO: 21 and a VL domain comprising the amino acid sequence of SEQID NO:
 22. 26. The method of claim 25, further comprising the step ofadministering a Factor D antagonist, wherein the Factor D antagonist isan anti-Factor D antibody.
 27. The method of claim 26, wherein theanti-Factor D antibody is lampalizumab.
 28. A method of treating wet AMDin a human subject in need thereof, the method comprising administeringto the human subject a therapeutically effective amount of an antibodythat specifically binds HtrA1, wherein the antibody comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 21 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 22. 29. Themethod of claim 28, further comprising the step of administering aFactor D antagonist, wherein the Factor D antagonist is an anti-Factor Dantibody.
 30. The method of claim 29, wherein the anti-Factor D antibodyis lampalizumab.
 31. A method of treating geographic atrophy in a humansubject in need thereof, the method comprising administering to thehuman subject a therapeutically effective amount of an antibody thatspecifically binds HtrA1, wherein the antibody is a full-length antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 160 and a light chain comprising the amino acid sequence of SEQ IDNO:
 159. 32. The method of claim 31, further comprising the step ofadministering a Factor D antagonist, wherein the Factor D antagonist isan anti-Factor D antibody.
 33. The method of claim 32, wherein theanti-Factor D antibody is lampalizumab.
 34. A method of treating wet AMDin a human subject in need thereof, the method comprising administeringto the human subject a therapeutically effective amount of an antibodythat specifically binds HtrA1, wherein the antibody is a full-lengthantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 160 and a light chain comprising the amino acid sequence ofSEQ ID NO:
 159. 35. The method of claim 34, further comprising the stepof administering a Factor D antagonist, wherein the Factor D antagonistis an anti-Factor D antibody.
 36. The method of claim 35, wherein theanti-Factor D antibody is lampalizumab.
 37. A combination therapycomprising: (a) an antibody that specifically binds HtrA1, wherein theantibody comprises a binding domain comprising the following six HVRs:(i) HVR-H1 comprises the amino acid sequence of DSEMH (SEQ ID NO: 7);(ii) HVR-H2 comprises the amino acid sequence of GVDPETEGAAYNQKFKG (SEQID NO: 8); (iii) HVR-H3 comprises the amino acid sequence of GYDYDYALDY(SEQ ID NO: 3); (iv) HVR-L1 comprises the amino acid sequence ofRASSSVEFIH (SEQ ID NO: 9); (v) HVR-L2 comprises the amino acid sequenceof ATSNLAS (SEQ ID NO: 10); and (vi) HVR-L3 comprises the amino acidsequence of QQWSSAPWT (SEQ ID NO: 11); and (b) a Factor D antagonist,wherein the Factor D antagonist is an anti-Factor D antibody.
 38. Thecombination therapy of claim 37, wherein the anti-Factor D antibody islampalizumab.
 39. A combination therapy comprising: (a) an antibody thatspecifically binds HtrA1, wherein the antibody comprises a bindingdomain comprising the following six HVRs: (i) an HVR-H1 comprising theamino acid sequence of DSEX₁H (SEQ ID NO: 1), wherein X₁ is Met or Leu;(ii) an HVR-H2 comprising the amino acid sequence of GVDPETX₂GAAYNQKFKG(SEQ ID NO: 2), wherein X₂ is Glu or Asp; (iii) an HVR-H3 comprising theamino acid sequence of GYDYDYALDY (SEQ ID NO: 3); (iv) an HVR-L1comprising the amino acid sequence of RASSSVX₃FIH (SEQ ID NO: 4),wherein X₃ is Glu or Asn; (v) an HVR-L2 comprising the amino acidsequence of ATSX₄LAS (SEQ ID NO: 5), wherein X₄ is Asn, His or Glu; and(vi) an HVR-L3 comprising the amino acid sequence of QQWX₅SX₆PWT (SEQ IDNO: 6), wherein X₅ is Ser or Tyr and X₆ is Ala or Asn; and (b) a FactorD antagonist, wherein the Factor D antagonist is an anti-Factor Dantibody.
 40. The combination therapy of claim 39, wherein theanti-Factor D antibody is lampalizumab.
 41. A combination therapycomprising: (a) an antibody that specifically binds HtrA1, wherein theantibody comprises a VH domain comprising the amino acid sequence of SEQID NO: 21 and a VL domain comprising the amino acid sequence of SEQ IDNO: 22; and (b) a Factor D antagonist, wherein the Factor D antagonistis an anti-Factor D antibody.
 42. The combination therapy of claim 41,wherein the anti-Factor D antibody is lampalizumab.
 43. A combinationtherapy comprising: (a) an antibody that specifically binds HtrA1,wherein the antibody comprises a heavy chain amino acid sequence of SEQID NO: 160 and a light chain amino acid sequence of SEQ ID NO: 159; and(b) a Factor D antagonist, wherein the Factor D antagonist is ananti-Factor D antibody.
 44. The combination therapy of claim 43, whereinthe anti-Factor D antibody is lampalizumab.